CN1845995A - Chimeric antigens for breaking host tolerance to foreign antigens - Google Patents

Abstract

This invention discloses a composition and method for inducing an immune response against an antigen. In particular, the composition and method induce an immune response against a foreign antigen recognized as a "self" antigen by the host, thereby disrupting the host's tolerance to these antigens. A chimeric antigen comprising an immune response domain and a target-binding domain, wherein the target-binding domain includes an antibody fragment, is presented to the host's immune system, enhancing the immune response against the foreign antigen or the tolerant antigen. The antigen-presenting cell takes up, processes, and presents the chimeric antigen, inducing both humoral and cellular immune responses against the desired antigen.

Description

Chimeric antigens for breaking host tolerance to foreign antigens

related application

This application claims priority to US Provisional Application No. 60/493,449, filed August 8, 2003, the contents of which are hereby incorporated by reference.

technical field

The present invention relates to methods and compositions for eliciting or enhancing an immune response (immune response) and for breaking host tolerance to foreign antigens.

Background technique

When a healthy host (human or animal) encounters a foreign antigen, such as a protein derived from bacteria, viruses and/or parasites, the host typically mounts an immune response. The immune response may be a humoral immune response and/or a cellular immune response. In a humoral immune response, antibodies are produced by B cells and secreted into the blood and/or lymph in response to antigenic stimulation. The antibody then neutralizes the antigen (such as a virus) by specifically binding to the surface of the antigen, labeling the antigen so that the antigen is destroyed by phagocytic and/or complement-mediated mechanisms, or by blocking binding or by Enhances the clearance of free antigens from circulation. The cellular immune response is characterized by the selection and increase of specific helper T lymphocytes and cytotoxic T lymphocytes capable of directly or indirectly clearing cells containing the antigen.

In some individuals, the immune system fails to mount a response to certain foreign antigens. When antigens do not prompt the production of specific antibodies and/or killer T cells, the immune system cannot prevent ensuing disease. Thus, an infectious agent, such as a virus, can develop a chronic infection and the host's immune system becomes tolerant to the antigens produced by the infectious agent.

Improper presentation of foreign antigens to the host's immune system may be a contributing factor in the development of chronic infection, as the mechanism by which the infectious agent evades the host's immune system has not been clearly established. Antigen presenting cells (APCs) process encountered antigens in different ways depending on the location of the antigen. Exogenous antigens are endocytosed and subsequently processed within the endosomes of the antigen-presenting cell. Peptide fragments generated from the exogenous antigen are complexed with class II major histocompatibility complex (MHC) and presented on the surface of the cell. This complex is presented to CD4 ⁺ T cells which stimulate CD4 ⁺ T helper cells to secrete cytokines which can stimulate B cells to produce antibodies against the exogenous antigen (humoral response). On the other hand, intracellular antigens are processed and presented in complex with MHC class I on the surface of antigen-presenting cells. Antigens are presented to CD8 ⁺ T cells causing cytotoxic T cells (CTLs) to mount an immune response against host cells bearing the antigen.

In patients with chronic viral or parasitic infections (where the organism resides within the host cell at some point in its life cycle), the host cell will produce and express the antigen within it, and there will be secreted antigen. For example, in the blood of chronic human hepatitis B virus (HBV) carriers, virions, HBV surface and core antigen surrogates (in the form of e-antigens) will be detected.

Effective treatment of chronic infection requires strong CTL responses against antigens associated with the infectious agent. This can be achieved by eliciting a cellular response by generating the antigen within the host cell or by delivering the antigen to the appropriate cellular compartment so that it can be processed and presented. Various routes of intracellular delivery of antigens have been reported in the literature. Among them, the use of viral vectors (Lorenz et al., Hum. Gen. Ther. 10: 623-631 (1999)), cDNA transfected cells (Donnelly et al., Ann. Rev. Immunol. 15: 617 (1997) )) and expression of antigens by injection of cDNA vectors (Lai et al., Crit. Rev. Immunol. 18: 449-484 (1998); and US Pat. No. 5,589,466) have been reported in the literature. In addition, DNA vaccines expressing antigens against dendritic cells have also been reported (You, et al., Cancer Res 61:3704-3711 (2001)).

Delivery vehicles capable of carrying antigens to the cytoplasmic compartment of cells for processing by the MHC class I pathway have also been utilized. The use of adjuvants for the same purpose has been described in detail by Hilgers, et al. (Vaccine 17:219-228 (1999)). Another approach is the cytoplasmic delivery of antigens using biodegradable microspheres, which has been demonstrated by the generation of Th1 immune responses against ovalbumin peptides (Newman, et al., J Control Release 54:49-59 (1998) and Newman, et al., J Biomed Mater Res 50:591-597 (2000)). In addition, antigen-presenting cells, such as dendritic cells, take up PLGA nanospheres (Newman, et al., J Biomed Mater Res 60:480-486 (2002)).

The ability of dendritic cells to capture, process and present antigens and to stimulate naïve T cells has made them very important tools in the development of therapeutic vaccines (Laupeze, et al., Hum Immunol 60:591-597 (1999)). Targeting antigen to dendritic cells is a critical step in antigen presentation and for this purpose a number of receptors present on dendritic cells of the Fc region of antibodies have been developed (Regnault, et al., J Exp Med 189:371 -380 (1999)). Additional examples of this pathway include ovarian cancer Mab-B43.13, anti-PSA antibodies, and anti-HBV antibody-antigen complexes (Wen, et al., Int Rev Immunol 18:251-258 (1999)). Cancer immunotherapy using dendritic cells loaded with tumor-associated antigens has been shown to generate tumor-specific immune responses and antitumor activity (Fong and Engleman, Ann Rev Immunol 96:1865-1972 (2000); and Campton, et al. J Invest Dermatol 115:57-61 (2000)). The use of tumor-antigen-pulsed dendritic cells has achieved good results in clinical experiments in vivo (Tarte and Klein, Leukemia 13: 653-663 (1999)). These studies clearly demonstrate the efficacy of using dendritic cells to generate immune responses against cancer antigens.

Antigen presentation can also be affected by mannose receptors on antigen-presenting cells instead of or in conjunction with Fc receptors. Macrophage mannose receptor (MMR), also known as CD206, is expressed on antigen presenting cells such as dendritic cells (DC). This molecule is a member of the C-type lectin family of endocytic receptors. Mannosylated antigens can be attached and internalized by CD206. It is generally believed that exogenous antigens are mainly processed and presented through the MHC class II pathway. However, when targeting via CD206, there is evidence that both the MHC class I and MHC class II pathways are involved (Apostolopoulos et al., Eur. J Immunol. 30:1714 (2000); Apostolopoulos and McKenzie, Curr. Mol. Med. 1: 469 (2001); Ramakrishna et al., J Immunol. 172: 2845-2852 (2004)).

Infectious diseases and cancer are major public health concerns. For example, statistics from the World Health Organization indicate that more than 2 billion people are infected with HBV. Of these, 370 million are chronically infected and therefore at high risk of developing cirrhosis and hepatocellular carcinoma. About 170 million people worldwide are chronic carriers of HCV, for which there is no effective preventive or therapeutic vaccine.

According to the World Health Organization, 10 million people are diagnosed with cancer every year. Cancer kills 6 million people every year, accounting for 12% of deaths worldwide. Accordingly, there is a need for novel, therapeutically effective compositions and methods that can elicit immune responses against infection and cancer, as well as new methods for producing such compositions.

Contents of the invention

The present invention provides a chimeric antigen for eliciting an immune response, the chimeric antigen comprises an immune response domain (immune response domain) and a target binding domain (targetbinding domain), wherein the target binding domain comprises an antibody fragment.

Another aspect of the invention provides a method of enhancing antigen presentation in an antigen presenting cell, the method comprising contacting the antigen presenting cell with a composition comprising a chimeric antigen of the invention.

Another aspect of the present invention also provides a method of activating an antigen-presenting cell, the method comprising contacting the antigen-presenting cell with the chimeric antigen of the present invention.

One aspect of the invention provides a method of eliciting an immune response comprising administering to a patient a composition comprising a chimeric antigen of the invention.

Another aspect of the invention provides a method of breaking tolerance comprising administering to a patient a chimeric antigen of the invention. In preferred embodiments, the patient is chronically infected with a virus or obligate intracellular parasite.

One aspect of the invention provides a method of treating an immuno-treatable disease comprising administering to a patient in need thereof a therapeutically effective amount of a chimeric antigen of the invention. In a preferred embodiment, the immuno-treatable disease is an infection, especially a chronic infection or cancer.

Another aspect of the invention also provides a method of vaccinating a subject against infection, the method comprising administering to the subject a chimeric antigen of the invention. Subjects can be vaccinated prophylactically or therapeutically. In preferred embodiments, the subject is immunoreactive to more than one epitope of the chimeric antigen, and more preferably, to more than one epitope of the immunoreactive region. Preferably, the infection is a viral infection or an obligate intracellular parasitic infection.

Another aspect of the present invention provides a pharmaceutical composition, which comprises the chimeric antigen of the present invention and a pharmaceutically acceptable excipient.

One aspect of the invention provides articles of manufacture comprising a chimeric antigen of the invention and instructions for administering the chimeric antigen to a patient in need thereof.

Another aspect of the present invention provides a polynucleotide encoding a chimeric antigen, the polynucleotide comprising a first polynucleotide encoding an immune response domain and a second polynucleotide encoding a target binding domain. A polynucleotide moiety, wherein the target binding domain comprises an antibody fragment. The invention also provides microorganisms and cell lines comprising such polynucleotides.

Another aspect of the present invention also provides a method for generating the chimeric antigen of the present invention, the method comprising providing a microorganism or cell line (the microorganism or cell line comprising a polynucleotide encoding the chimeric antigen of the present invention), and The microorganism or cell line is grown under conditions capable of expressing the chimeric antigen.

One or more embodiments of the present invention will be described in detail below with reference to the drawings and description. Other features, objects, and advantages of the invention will be apparent from the description, drawings, and claims.

Description of drawings

Figure 1A provides a schematic diagram of the structure of a chimeric antigen of the invention as a monomer, wherein the chimeric antigen has two parts, an immune response domain and a target binding domain. The schematic also shows the preferred embodiment, where a hinge region is present. Figure 1B provides a schematic diagram of the structure of the chimeric antigen of the present invention in its normal state, ie assembled as a dimer. The schematic shows a particularly preferred embodiment wherein the chimeric antigen comprises a 6xHis tag and a linker peptide in addition to the immune response domain and target binding domain.

Figure 2 shows that T cells are stimulated with HBV S1/S2-TBD to generate a cytotoxic T cell (CTL) response specific to an epitope from the HBV S1 protein.

Figure 3 shows stimulation of T cells with HBV S1/S2-TBD to generate cytotoxic T cell (CTL) responses specific to epitopes from the HBV S2 protein.

Figure 4 shows the comparison of the uptake of HBVS1/S2-TBD, IgG1 and IgG2 by mature dendritic cells as a function of concentration.

Figure 5 shows the correlation between the binding of HBV S1/S2-TBD and the expression of CD32 on dendritic cells and the correlation between the expression of CD206.

Detailed ways

A. Overview

Compositions and methods for eliciting an immune response against an antigen are disclosed. In particular, the compounds and methods can elicit an immune response against foreign antigens that the host perceives as "self" antigens, thereby breaking host tolerance to these antigens. Presentation to the host's immune system of a chimeric antigen comprising an immune response domain and a target binding domain (where the target binding domain comprises an antibody fragment) enhances the immune response against a foreign antigen or a tolerant antigen. Antigen-presenting cells take up, process, and present chimeric antigens, eliciting humoral and cellular immune responses against the desired antigen.

b. Definition

Before describing the present invention in further detail, unless otherwise specified, the terms used in this application are defined as follows.

"Antibody" refers to an immunoglobulin molecule with a specific amino acid sequence produced by B lymphocytes caused by an antigen (immunogen) in humans or other animals. These molecules are characterized by reacting specifically with that antigen, each defined in terms of the other.

"Antibody response" or "humoral response" refers to a type of immune response in which antibodies are produced by B lymphocytes and secreted into the blood and/or lymph in response to antigenic stimulation. In a normal functional immune response, the antibody specifically binds an antigen on the surface of a cell (eg, a pathogen), marking the cell for destruction by phagocytotic cells and/or complement-mediated mechanisms. Antibodies can also circulate systemically and bind free virions. Binding of the antibody neutralizes the virion and prevents it from infecting cells, and marks the virion for clearance from circulation by phagocytosis or filtration in the kidney.

"Antigen" means any substance that enters the body and comes into contact with appropriate cells, resulting in a state of sensitization and/or immune response, which can in a demonstrable manner in vivo or in vitro interact with a sensitized subject's antibodies and/or or immune cell response.

"Antigen presenting cell" refers to an auxiliary cell in the process of antigen induction, whose function is mainly to process and present antigen to lymphocytes. The interaction of antigen presenting cells with antigens is a critical step in the induction of immunity as it enables lymphocytes to encounter and recognize antigenic molecules and activates the lymphocytes. Exemplary antigen-presenting cells include macrophages, Langerhans-dendritic cells, nodular dendritic cells (follicular dendritic-like cells), and B cells.

"B cell" refers to a lymphocyte capable of producing immunoglobulins or antibodies that interact with an antigen.

" _CH1 region" refers to the region of the heavy chain constant domain on an antigen-binding fragment of an antibody.

"Cellular response" refers to an immune response mediated by specific helper T cells and killer T cells, and the killer T cells can directly eliminate virus-infected cells or cancer cells.

As used herein, the term "chimeric antigen" refers to a polypeptide that includes an immune response domain and a target binding domain. The immune response domain and target binding domain can be directly or indirectly linked covalently or non-covalently.

"Cytotoxic T lymphocytes" are specialized lymphocytes capable of destroying foreign cells and host cells infected with infectious agents capable of producing viral antigens.

"Epitope" refers to the simplest form of an antigenic determinant on a complex antigen molecule; it is a specific portion of an antigen that is recognized by an immunoglobulin or T cell receptor.

"Fusion protein" refers to a protein formed by the expression of a hybrid gene composed of the combination of two or more gene sequences.

"Hinge region" refers to the portion of an antibody that connects the Fab fragment to the Fc fragment; the hinge region contains the disulfide bonds that covalently link the two heavy chains.

The term "homologue" refers to a molecule that exhibits homology to another molecule by, for example, having identical or similar sequences of chemical residues at corresponding positions. The term "% homologous" or "% homologous" refers to the percentage of identical or similar nucleotides or amino acids at the same position in homologous polynucleotides or polypeptides. For example, if 75 out of 80 residues in two proteins are identical, then the two proteins are 93.75% homologous. There are various software programs known to those skilled in the art to determine percent homology.

"Host" refers to a warm-blooded animal, including humans, suffering from an immuno-treatable disease, such as infection or cancer. As used herein, "host" also refers to a warm-blooded animal, including humans, to which a chimeric antigen is administered.

In the context of the present invention, "hybridization" refers to the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred pairing mechanism involves hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases) of strands of oligomeric compounds, which hydrogen bonding can be Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reverse Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds. Hybridization can occur under different circumstances. The term "hybridization" and the like in the context of polynucleotides refer to common hybridization conditions, preferably hybridization under 50% formamide/6X SSC/0.1% SDS/100 μg/ml mDNA conditions, Wherein the temperature for hybridization is higher than 37°C and the temperature for washing in 0.1X SSC/0.1% SDS is higher than 55°C.

"Immunity" or "immune response" refers to the body's response to an antigen. In particular embodiments, it refers to the ability of an organism to resist or protect itself from infectious disease.

"Immune Response Domain (IRD)" refers to the distinct conformational antigenic portion of a bifunctional chimeric antigenic molecule. The immune response domain includes one or more antigens and/or one or more recombinant antigens.

As used herein, the term "immuno-treatable disease" refers to a disease or condition that can be prevented, suppressed or alleviated by eliciting or modulating an immune response in a patient.

"Lymphocytes"refers to the subclass of nucleated cells present in the blood that mediate specific immune responses.

"Monoclonal antibody" or "mAb" refers to antibodies produced by fused hybrid cells, ie, clones or genetically homogeneous populations of hybridoma cells. Hybrid cells are cloned in order to establish cell lines that produce specific monoclonal antibodies that are chemically and immunologically homologous, ie, that recognize only one type of antigen.

"Peptide bond" refers to two or more amino acids covalently linked by a substituted amide bond between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid.

"Pharmaceutically acceptable" means a non-toxic composition that is physiologically compatible with humans or other animals.

"Pharmaceutically acceptable excipients" include substances that are nontoxic and physiologically compatible with humans or other animals, such as adjuvants, carriers, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives agents and their analogues.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides, of any length. The term refers only to the primary structure of the molecule. Thus, the term includes both double- and single-stranded DNA and RNA. It also includes known types of modifications of one or more naturally occurring nucleotides and analogs, for example, labeling, methylation, "capping", substitutions known in the art; internucleotide modifications, Such as modification with uncharged linkages (for example, methyl phosphate, phosphotriester, phosphate amide, carbamate (salt), etc.) and modification with charged linkages (for example, phosphorothioate, disulfide Phosphoesters, etc.), modifications that include accessory moieties, such as proteins (including, for example, ribozymes, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (such as acridine pyridine, psoralen, etc.) including chelating agents (e.g., metals, radiometals, boron, metal oxides, etc.), modifications involving alkylators, modifications with modified linkages (e.g., α-anomeric nucleic acids, etc.), and polynucleotides in unmodified form.

A "proteolytic cleavage site" refers to a proteolytic enzyme that hydrolyzes (breaks) a polypeptide chain.

"Stringent hybridization conditions" or "stringent conditions" as used herein refers to conditions under which a compound of the present invention hybridizes to its target sequence and hybridizes to a very small (minimum) amount of other sequences.

The term "subject" refers to any warm-blooded animal, preferably a human being.

"Tag" refers to a marker or marker sequence used to isolate or purify a molecule comprising the tag. Exemplary tags include 6xHis tags.

"T cell" refers to a class of lymphocytes responsible for antigen-specific cellular interactions that mediate humoral and cellular immune responses.

"Target binding domain (TBD)" refers to a protein comprising an antibody fragment capable of binding to a receptor on an antigen-presenting cell, especially a dendritic cell, and which is subsequently transported into the antigen-presenting cell by receptor-mediated uptake .

The term "therapeutically effective amount" refers to an amount of a chimeric antigen or a polynucleotide encoding a chimeric antigen, which is sufficient to elicit effective B cells, cytotoxic T lymphocytes (CTL) and/or helper cells against the antigen. A T lymphocyte (Th) response is sufficient to block or treat or at least partially suppress or alleviate symptoms and/or complications of a disease or disorder.

As used herein, the term "treatment" includes any treatment in which a chimeric antigen can be used to treat a disease in an animal, especially a human, and includes: (i) preventing the disease from occurring in a subject, who may be susceptible to the disease but A subject who has not been diagnosed with the disease; (ii) inhibiting the disease, eg, inhibiting or slowing its development; (iii) alleviating the disease, eg, causing remission of the disease or its symptoms.

As used herein, "heterotype" refers to originating from a species different from the host. For example, a recombinantly expressed antibody cloned from the murine genome is of the human but not murine isotype, regardless of whether the recombinantly expressed antibody was produced in bacterial, insect or murine cells.

C. Novel Chimeric Antigens

The invention provides a chimeric antigen for eliciting an immune response, the antigen comprising an immune response domain and a target binding domain, wherein the target binding domain comprises an antibody fragment. According to the invention, the chimeric antigen is, preferably, capable of binding to Fc receptors and/or macrophage mannose receptors. The antibody fragment may be heterotyped or non-heterotyped for the host.

In a preferred embodiment of the invention, the chimeric antigen is capable of eliciting a humoral and/or cellular immune response. A cellular immune response may include a Th1 response, a Th2 response, and/or a cytotoxic T lymphocyte (CTL) response. In another preferred embodiment, the chimeric antigen elicits a polyepitopic immune response. The multi-epitopic immune response may comprise a response to at least one epitope of an immune response domain and/or a response to at least one epitope of a target binding domain. Alternatively the polyepitopic response may be limited to a response to more than one epitope of the immune response domain.

The chimeric antigen of the present invention includes two parts, an immune response domain comprising an antigenic sequence (such as a viral antigen), and a target binding domain comprising an antibody fragment ( FIG. 1 ). In preferred embodiments, the immune response domain may be linked to the target binding domain by any method known to those skilled in the art. Linkage means for linking the immune response domain to the target binding domain can include, but are not limited to, covalent peptide bonds, chemical linkages, leucine zippers, and biotin/avidin. In preferred embodiments, the immune response domain and target binding domain are cloned as a single fusion protein. The covalent peptide linkages of the fusion protein may include other peptide sequences such as SRPQGGGS or VRPQGGGS (SEQ ID NO: 1). In another preferred embodiment, the various immune response domains are biotinylated and the target binding domains are generated with streptavidin as a fusion protein in order to facilitate the generation of multiple chimeric antigens. Alternatively, each of the immune response domain and target binding domain can be expressed as a fusion to a leucine zipper moiety which will result in the binding of the two parts of the chimeric antigen by mixing. Ultimately, the immune response domain and target binding domain can be expressed independently and then chemically combined by methods known to those skilled in the art. An exemplary method involves covalently linking the two domains using a protein cross-linker such as dimethyl subermidate.

The immune response domain essentially provides the antigenic portion of the chimeric antigen. The immune response domain includes at least one antigenic portion of an integral body required for an immune response. The chimeric antigen, optionally, can include more than one immune response domain. In preferred embodiments, the immune response domain comprises at least one antigenic portion of an infectious agent such as a virus or obligate intracellular parasite or at least one antigenic portion of a cancer antigen. More preferably, the immune response domain includes at least one antigenic portion of an infectious virus.

Examples of preferred infectious viruses include: Retroviridae (e.g. human immunodeficiency virus, such as human immunodeficiency virus-1 (HIV-1), also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolated viruses such as HIV-LP); picornaviridae (eg, poliovirus, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, eikeviruses) ; Calciviridae (eg, strains that cause gastroenteritis); Envelopeviridae (eg, equine encephalitis virus, rubella virus); Flaviridae (eg, hepatitis C virus, dengue virus , encephalitis virus, yellow fever virus); Coronaviridae (eg, Coronaviridae); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Filoviridae (eg, Ebola virus); Paramyxoviruses Viridae (e.g., parainfluenza, mumps, measles, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza); Bunyaviridae (e.g., hantaan, bunga, arenaviridae (hemorrhagic fever viruses); reoviridae (such as reoviruses, orbiviruses, and rotaviruses); bisRNAviridae; hepadnaviridae (human B Hepatitis virus (HBV), duck hepatitis B virus (DHBV)); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpes Viridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), Epstein-Barr virus, herpes virus); Poxviridae (variola virus, vaccinia virus, pox virus); and iridoviruses family (eg African swine fever virus); and unclassified viruses (eg delta hepatitis species, non-A non-B hepatitis species (category 1 - internal transmission); category 2 - parenteral transmission; Norwalk virus and related viruses, and astrovirus). In some embodiments of the present invention, the immune response domain of the chimeric antigen comprises at least one antigenic portion of one or more proteins selected from the group consisting of: HBV protein, DHBV protein and HCV protein. Particularly preferred HBV proteins for use in the present invention include, but are not limited to, HBV S1/S2, HBV S1/S2/S, HBV core, HBV core ctm (C-terminal modification), HBV e antigen, and HBV polymerase. Particularly preferred DHBV proteins for use in the present invention include, but are not limited to, DHBV PreS/S, DHBVPreS, DHBV core, and DHBV polymerase. Particularly preferred HCV proteins for use in the present invention include, but are not limited to, HCV core (1-191), HCV core (1-177), HCV E1-E2, HCV E1, HCV E2, HCV NS3, HCV NS5A and NS4A . Other preferred viral antigens for use in the present invention include HIV gp120, HSV alkaline nuclease, and human papillomavirus (HPV) capsid proteins L1 and L2, and early domain proteins HPV E1, HPV E2, HPV E4, HPV E5, HPV E6 and HPV E7.

Examples of preferred obligate intracellular parasites include: Tetrahymena (e.g. Tetrahymena piriformis), Plasmodium (e.g. Plasmodium falciparum), Cryptospiridium sp., Spraguea sp. (eg, S. lophii), Giardia, Toxoplasma (eg, T. gondii, Trypanosoma cruzi), Leishmania, Rickettsia (eg, Rickettsia prauszii), Chlamydia, Clade Bacillus (eg, Mycobacterium tuberculosis), Legionella, Listeria (eg, Listeria monocytogenes), Coxella (eg, Coxella burgdorferi), Shigella, Ehrlichia , and Bartonelia sp. Preferred cancer antigens include: prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), MUCl, CA125, WT1, Her-2/neu, carcinoembryonic antigen (CEA), MAGE-3, MART-1, gp100, NY-ESSO-1, CA19.9, TAG72, CA15.3, CA27.9, gp120, prostatic acid phosphatase (PAP), heat shock protein, alpha-fetoprotein (AFP), telomerase, and NET state activating system (ras).

In another embodiment of the invention, the immune response domain of the chimeric antigen comprises a 6xHis tag fused to one or more antigen moieties.

According to the invention, the chimeric antigen is a protein capable of binding to Fc receptors and/or CD206 on antigen-presenting cells, especially dendritic cells, and is subsequently transported to the antigen by receptor-mediated uptake presented in the cell. According to the invention, the presence of antibody fragments enhances the uptake of the chimeric antigen via Fc receptors on antigen presenting cells, especially dendritic cells. With this specific binding and internalization, viral antigens are processed and presented as foreign antigens. An immune response against previously tolerated antigens can thus be efficiently elicited. The target binding domain includes antibody fragments that are either heterotyped or non-heterotyped to the host. In a preferred embodiment of the invention, the antibody fragment comprises a murine Fc fragment. In a more preferred embodiment of the present invention, the target binding domain comprises an Fc fragment, a hinge region, part of the _CH1 region, and the chimeric antigen comprises a protein suitable for linking the target binding domain to the immune response domain. peptide bond. In a more preferred embodiment, the target binding domain comprises an immunoglobulin heavy chain fragment, optionally further comprising a hinge region. In particularly preferred embodiments, the heavy chain fragment comprises amino acids, VDKKI of the _CH1 domain (SEQ ID NO: 2) and/or part or all of the _CH2 and _CH3 domains.

As described above, antigens ligated and internalized by CD206 can be presented by MHC class I and MHC class II, thus eliciting cellular and humoral immune responses. Thus, in preferred embodiments, the chimeric antigen is glycosylated. The immune response domain and/or target binding domain may be glycosylated. In particularly preferred embodiments, the chimeric antigen may be mannose glycosylated by hypermannose glycosylation or by pauci mannoseglycosylation (Jarvis, Viurlogy 310:1 -7(2003)).

D. Novel Approaches Using Chimeric Antigens

The invention includes methods of eliciting an immune response comprising administering to a subject a composition comprising a chimeric antigen of the invention.

In order to provide efficient presentation of this antigen, the inventors developed a novel chimeric antigen comprising an immune response domain and a target binding domain, wherein the target binding domain comprises an antibody fragment. While not limited to a particular theory of the invention, the molecule is captured by antibody fragments that bind to specific receptors on antigen-presenting cells, while viral antigens are processed and complexed with class I and II major histocompatibility complexes (MHC). present. Such processing and antigen presentation by MHC class I elicits an enhanced cytotoxic T lymphocyte response, leading to the clearance of any infectious agent associated with that antigen of the immune response domain. In addition, antigen presentation by MHC class II molecules elicits a humoral response that also facilitates clearance of the antigen from infected cells and/or from circulation.

The invention also includes a method of breaking tolerance comprising administering a chimeric antigen of the invention to a subject. Antigens that are processed as "self" in chronic infection are recognized as "foreign" antigens during the presentation of antigens to elicit cellular and/or humoral immune responses by means of chimeric antigens. Thus, the host's immune system will mount a CTL response to clear the infected cells. Simultaneously, in response to the chimeric antigen, the antibodies elicited will bind to the infectious agent and clear it from circulation or block the binding of the infectious agent to host cells. Accordingly, the present invention is designed to generate chimeric antigens capable of eliciting a broad immune response in a target subject suffering from a chronic infection that is tolerated by the host's immune system. In preferred embodiments, the chimeric antigen breaks tolerance to the antigen in a subject, wherein the subject is chronically infected with an infectious agent such as a virus or parasite or has cancer. More preferably, the infectious agent resides within the host cell at some point in its life cycle.

In preferred embodiments, immunogens for preselected antigens not recognized or tolerated by the host immune system due to the presence of antibody fragments and the presence of glycosylation introduced in eukaryotic (eg, insect) cell expression systems Sex is enhanced. Due to the presence of antibody components and glycosylation, such chimeric antigens will bind to specific receptors present on various immune cell types including dendritic cells, macrophages, B cells, and granulocytes.

Yet another aspect of the invention provides a method of activating an antigen-presenting cell, the method comprising contacting the antigen-presenting cell with a chimeric antigen of the invention. The invention also provides a method of increasing antigen presentation in an antigen presenting cell, the method comprising contacting the antigen presenting cell with a composition comprising a chimeric antigen of the invention. The chimeric antigen can be contacted with antigen presenting cells, preferably dendritic cells, in vivo or in vitro. In preferred embodiments, contacting the chimeric antigen with an antigen presenting cell activates the antigen presenting cell and increases antigen presentation of more than one epitope. Such a multi-epitopic response may include presentation of one or more epitopes of an immune response domain and/or presentation of one or more epitopes of a target binding domain.

The invention also provides a method of treating an immuno-treatable disease comprising administering a therapeutically effective amount of a chimeric antigen of the invention to a subject in need thereof. In preferred embodiments, the immuno-treatable disease is infection or cancer. The infection can be a viral infection, a parasitic infection, or a bacterial infection. Preferably, the infection will involve a stage in which the infectious agent is present within the host cells. More preferably, the immuno-treatable disease is a chronic viral infection. More preferably, the immuno-treatable disease is chronic hepatitis B virus (HBV) infection or chronic hepatitis C virus (HCV) infection. For the treatment of HBV, the immune response domain preferably includes at least one antigenic portion of a protein selected from the group consisting of: HBV core protein, HBV S protein, HBV S1 protein, HBVS2 protein, and combinations thereof. For the treatment of HCV, the immune response domain preferably comprises at least one antigenic portion of a protein selected from the group consisting of HCV core (1-191) protein, HCV core (1-177) protein, HCV E1 protein, HCV E2 protein, HCV E1-E2 protein, HCV NS3A protein, HCV NS5A protein, and compositions thereof.

In preferred embodiments, administration of the chimeric antigen elicits a greater immune response than administration of the immune response domains alone. The extent of this immune response can be measured, for example, by (i) the amount of antigen-specific antibodies present in the subject of interest; (ii) interferon- The amount of γ, the antigen-presenting cell loaded with the chimeric antigen or only the immune response domain; or (iii) the amount of antigen-specific CD8 ⁺ T cells in the immune response elicited by exposure to the antigen-presenting cell, the antigen Presenting cells are loaded with the chimeric antigen or just the immune response domain.

The potency of the chimeric antigen can be assessed by presenting the chimeric antigen to dendritic cells to generate an immune response in vivo or in vitro. Dendritic cells process and present the chimeric antigen to T lymphocytes, which can be used as markers of T cell response to assess T cell proliferation and interferon-γ production. Specifically, in an in vitro setting, primitive dendritic cells are isolated and processed from surrounding blood. Activation of T cells via dendritic cells is assessed by measuring the levels of markers such as interferon-γ by known methods. See, eg, Berlyn, et al., Clin. Immunol 101(3):276-283 (2001). An increase in the percentage of T cells secreting at least 50% interferon-γ is predictive of an effect in vivo. In vivo, the chimeric antigen is directly parenterally introduced into a host in which available dendritic cells and other antigen-processing cells have the capacity to interact with the antigen and thereby process them.

Accordingly, the invention includes a method of vaccinating a subject against infection comprising administering to the subject a chimeric antigen of the invention. The subject can be vaccinated prophylactically or therapeutically. Preferably, the infection is a viral infection. The bifunctional nature of the molecule facilitates the targeting of antigens to antigen-presenting cells such as dendritic cells, making it a promising candidate for the treatment of chronic infectious diseases by targeting antibodies to antigen-presenting cells with the most potent stoichiometry of antigen unique approach. This is useful in the development of therapeutic vaccines for chronic viral infections such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, papillomavirus and herpes simplex virus, obligate intracellular parasites, and also Can be applied to all autologous antigens in diseases such as cancer and autoimmune diseases. Administration of these fusion proteins can elicit a broad range of immune responses from the host, including cellular and humoral immune responses. Thus, they can be used as therapeutic vaccines to treat subjects exhibiting immune tolerance to an existing infection, and as prophylactic vaccines to immunize subjects at risk of the spread of a particular infection.

E. Methods of Preparing Chimeric Antigens

One aspect of the invention provides a method for producing a chimeric antigen comprising (a) providing a microorganism or cell line, preferably a eukaryotic organism, more preferably a polynucleotide encoding a chimeric antigen is a non-mammalian microorganism or cell line; and (b) culturing said microorganism or cell line under conditions under which the chimeric antigen is expressed. Preferably, the microorganism or cell line is yeast, a plant cell line or an insect cell line. More preferably, the cell line is an insect cell line selected from Sf9, Sf21, Drosophila S2, High Five ^™ .

One embodiment of the invention utilizes established recombinant DNA techniques to generate fusion proteins of selected antigens and binding domains of interest necessary to practice the invention. The structure of the fusion protein is generated at the DNA level in conjunction with specific restriction sites, which are generated by introducing the desired DNA fragment into an expression vector and used to express the desired fusion protein in a heterologous expression system. The term "vector" as used herein refers to a plasmid capable of carrying DNA encoding a desired protein. Preferred plasmid vectors for use in the present invention include, but are not limited to, pFastBac HTa and the corresponding recombinant "bacmids" produced in DH10Bac ^™ E. coli (invitrogen).

The gene encoding the binding domain of interest can be obtained from any antibody-producing cell (eg, a monoclonal antibody-producing hybridoma cell) by polymerase chain reaction (PCR). To facilitate subsequent cloning steps, it is preferred to design oligonucleotide primers incorporating a unique restriction enzyme recognition site. Similarly, the antigenic portion of the immune response domain can be obtained from any cellular or viral RNA or DNA comprising a gene encoding the antigenic portion of the desired target. Preferably, PCR is used to obtain DNA encoding the antigenic portion of the immune response domain, and PCR primers are designed to incorporate unique restriction enzyme recognition sites for facilitating cloning. However, any recombinant DNA method can be used to obtain the DNA encoding the antigenic portion of the immune response domain. The polynucleotides encoding the target binding and immune response domains can then be combined in a single construct using standard cloning techniques. Alternatively, the separate domains can be cloned by means of DNA encoding linkers such as leucine zippers, streptavidin or biotinylation signals.

Preferably, the baculovirus system is used to express the chimeric antigens of the present invention, not only because of the large amount of heterologous protein produced, but also because of post-translational modifications of eukaryotic proteins that occur within infected insect cells, such as phosphorylation and glycosylation. Due to the difficulty of cloning directly in insect cells, it is preferred to generate the polynucleotide encoding the chimeric antigen in a bacterial system and transfer the final construct into a baculovirus/insect cell expression system. Transport systems such as the Bac-To-Bac ^™ system (Invitrogen) are known to those skilled in the art. The Bac-to-Bac ^™ system utilizes site-specific transposition via the bacterial transposon Tn7 for the transfer of relevant genes into the E. coli-insect cell shuttle vector (bacmid). The resulting recombinant bacmids are transfected into insect cells to generate baculoviruses expressing the recombinant proteins.

To generate baculoviruses, bacmids are transfected into insect cells such as Sf9 cells. After transfection, the cells are incubated for a period of time sufficient to expand the viral vector population. The medium containing the baculovirus was collected and stored at 4°C in the dark. The transfection can be demonstrated by examining the production of baculoviral DNA by subjecting the viral culture to PCR using primers specific for the desired DNA insert. Expression of the intracellular heterologous protein can be confirmed by any method known to those skilled in the art, such as SDS polyacrylamide gel electrophoresis (SDS-PAGE) or Western blotting.

Normalized multiplicity of infection (MOI) of recombinant bacmids was used to infect insect cells. Cells were seeded at a density of approximately 3×10 ⁵ cells/mL and incubated in suspension medium at 27.5° C. with shaking until the cell density reached approximately 2-3×10 ⁶ cells/mL. Standardized amounts of each recombinant baculovirus were then added to the cells. The incubation temperature was 27.5°C and the appropriate infection period was normalized for independent protein expression. The cells were harvested by centrifugation and used for purification of recombinant proteins. Unused portions of cells can be snap frozen in liquid nitrogen and stored at -70°C.

Chimeric antigens are preferably purified under denaturing conditions. Cells expressing the chimeric antigen are lysed with a denaturing buffer such as a buffer containing 6M guanidinium-HCl. Dissolution can be enhanced by mechanical means such as sonication. Lysates were centrifuged to remove unbroken cells and cellular debris. The supernatant was then loaded onto a Ni-NTA Super Flow (Qiagen) bead column pre-equilibrated with lysis buffer. After loading, the column is rinsed with a buffered denaturing solution (preferably comprising 6M guanidinium-HCl at a pH of approximately 8). At this point the denaturant can be exchanged for eg 8M urea in buffer solution. Lysis, loading and washing buffers preferably contain low concentrations (eg 1-40 mM) of imidazole. After buffer exchange, the column should be flushed with buffer until the _OD280 drops to eg <0.1. Bound protein can be eluted with a pH 8 buffer containing 8M urea and 250mM imidazole (elution buffer). Fragments containing the protein are pooled and dialyzed at 4°C against a low (eg 100 mM) salt denaturing dialysis buffer (preferably containing 8M urea) of complex changes. The dialyzed protein is then loaded onto an ion exchange column such as DEAE (diethylaminoethyl). In preferred embodiments, dithiothreitol (DTT) or other reducing agents are added to the protein prior to loading onto an ion exchange column. The chimeric antigen will pass through the DEAE column. Subsequently, the DEAE flow-through was collected and dialyzed stepwise against buffers containing decreasing concentrations of denaturant. In an exemplary method, the protein is then dialyzed against buffered 4M urea for at least 12 hours, then against buffered 2M urea for at least 12 hours, then against buffered 1M urea for at least 12 hours, then against buffered 0.5 Dialyzed against urea for at least 12 hours and finally against a buffer containing no denaturing agent for at least 12 hours, preferably followed by two further dialysis of 12 hours against fresh buffer containing no denaturing agent. The refolded protein can be purified and characterized by antibodies directed against different domains of the expressed protein using standard biochemical techniques including, eg, SDS gel electrophoresis, isoelectric focusing, or Western blot analysis.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, within the skill of the art. Such techniques are well described in the literature, see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ^Ed . , New York: Cold Spring Harbor Press, 1989; and Ausubel et al., Current Protocols in Molecular Biology , Wiley Interscience Publishers, (1995, as Supplemented April 2004, Supplement 66).

F. Novel Polynucleotides

Another aspect of the present invention provides a polynucleotide encoding a chimeric antigen, the polynucleotide comprising a first polynucleotide portion (part) encoding an immune response domain and a first polynucleotide encoding a target binding domain. The second polynucleotide portion (fraction). The first and second polynucleotide moieties can be on the same or different nucleotide strands.

The present invention provides a polynucleotide (preferably in an independent form) corresponding to or complementary to the coding sequence of the chimeric antigen, mRNA, and/or the coding sequence, and the polynucleotide includes the coding chimeric antigen variant protein (variant protein) ) polynucleotides; DNA, RNA, DNA/RNA hybrids, and related molecules; polynucleosides that are complementary or have at least 90% homology to a gene or mRNA sequence encoding a chimeric antigen or a portion thereof acid or oligonucleotide; and a polynucleotide or oligonucleotide that hybridizes to a gene encoding a chimeric antigen, mRNA, or to a polynucleotide encoding a chimeric antigen.

In addition, the present invention includes analogs of the genes encoding the chimeric antigens specifically disclosed herein. Analogs include, for example, mutants that retain the ability to elicit an immune response and preferably have at least 80%, more preferably 90%, and most preferably 95% identity to any polynucleotide encoding a chimeric antigen. Origin, the polynucleotide encoding the chimeric antigen is specifically described as SEQ ID Nos: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 44. Typically, such analogs differ by only 1 to 15 codons. Examples include polypeptides having minor amino acid changes, especially conservative amino acid substitutions, compared to the native amino acid sequence of a viral antigen or antibody fragment. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Amino acids encoded by genes are usually divided into four families: (1) acidic amino acids = aspartic acid, glutamic acid; (2) basic amino acids = lysine, arginine, histidine; (3) non-polar amino acids Sexual amino acids = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids = glycine, aspartame Amide, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids. For example, it is reasonable to expect the sole replacement of leucine by isoleucine or valine, the sole replacement of aspartic acid by glutamic acid, the sole replacement of threonine by serine, or the sole replacement of amino acids by structural Similar conservative substitutions of related amino acids will not have a major effect on biological activity. Having substantially the same amino acid sequence as any polypeptide disclosed in any of SEQ ID Nos: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 but with minor amino acid substitutions ( It does not substantially affect the ability of the chimeric antigen to elicit an immune response) polypeptide molecules having SEQ ID Nos: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 respectively within the definition of a chimeric antigen of the sequence described above. Derivatives include aggregate conjugates with other chimeric antigen molecules and covalent conjugates with unrelated chemical moieties. Covalent derivatives are prepared by methods known in the art by linking functionality to groups found within the amino acid chain of the chimeric antigen or to residue groups at the N- or C-terminus.

Abbreviations for amino acids are provided in Table 1.

Table 1: Abbreviations for Amino Acids Alanine Ala A arginine Arg R Asparagine Asn N aspartic acid Asp D. cysteine Cys C glutamic acid Glu E. Glutamine Gln Q Glycine Gly G Histidine His h Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine met m Phenylalanine Phe f proline Pro P serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Conservative amino acid substitutions can be made within a protein without altering the protein's conformation or function. Proteins of the invention may include 1 to 15 conservative substitutions. Such changes include: replacing any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; replacing aspartic acid (D) to glutamic acid (E) and vice versa; glutamine (Q) to asparagine (N) and vice versa; and serine (S) to threonine (T ),vice versa. Other substitutions are also considered conservative substitutions, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). The relatively hydrophobic methionine (M) is often interchangeable with leucine, isoleucine, and sometimes valine. Lysine (K) and arginine (R) are usually interchangeable at positions where the important characteristic of the amino acid residue is its charge, and the different pK of the two amino acid residues is not important of. Still other exchanges may be considered "conserved" in certain circumstances (see, e.g., Biochemistry 4 ^th Ed. , Lubert Stryer ed. (WH Freeman and Co.), pages 18-23; Henikoff and Henikoff, Proc Nat'l Acad Sci USA 89: 10915-10919 (1992); Lei et al., J Biol Chem 270(20): 11882-6 (1995)).

The present invention also provides a polynucleotide that hybridizes to a polynucleotide encoding a chimeric antigen, preferably under stringent conditions, such as SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 44 are described in detail. Stringent conditions for hybridization reactions are readily determined by one of ordinary skill in the art and are generally considered empirically based on probe length, wash temperature, and salt concentration. Typically longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured nucleic acid sequences to anneal again when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizing sequence, the higher the relative temperature to be used. Thus, it follows that higher relative temperatures will result in more stringent conditions for the reaction, while lower temperatures will result in more relaxed conditions for the reaction. For more details and descriptions of stringent conditions for hybridization reactions see, eg, Ausubel et al., supra, at pages 2.9.1-2.10.8 and 4.9.1-4.9.13.

The "stringent conditions" or "highly stringent conditions" defined here refer to but are not limited to those conditions: (1) low ionic strength and high temperature are used for washing, such as 0.015M sodium chloride/0.0015M sodium citrate /0.1% sodium dodecyl sulfate, 50°C; (2) use denaturants such as formamide during hybridization, for example, 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer pH6.5 with 750mM sodium chloride, 75mM sodium citrate, 42°C; or (3) 50% formamide, 5X SSC (0.75M NaCl, 0.075M sodium citrate ), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate, 42°C, and a wash in 0.2X SSC (sodium chloride/sodium citrate) at 42°C and a wash in 50% formamide at 55°C, followed by a highly stringent wash with 0.1X SSC containing EDTA at 55°C. "Conditions of moderate stringency" refer to, but are not limited to, those of Sambrook et al. supra, and include the use of wash solutions and hybridization conditions (eg, temperature, ionic strength, and % SDS) that are less stringent than those described above. An example of moderately stringent conditions refers to overnight incubation at 37°C in a specific solution comprising: 20% formamide, 5X SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5X Denhardt 's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, and then wash the filter with 1X SSC at about 37-50°C. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

Examples of polynucleotides encoding chimeric antigens include: any one sequence selected from SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 A polynucleotide encoding a chimeric antigen, or a nucleotide sequence of a chimeric antigen selected from any one of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, 46 and 48 , where T can optionally be U; for example, examples of nucleotides for chimeric antigens include, but are not limited to:

(a) Polynucleotides comprising or consisting of the following sequences: SEQ ID NOs: nucleotides 1 to 1326 of SEQ ID NO: 26, nucleotides 1 to 2004 of SEQ ID NO: 28, Nucleotides 1 to 1350 of SEQ ID NO: 30, Nucleotides 1 to 1293 of SEQ ID NO: 32, Nucleotides 1 to 1794 of SEQ ID NO: 34, SEQ ID NO: 36 nucleotides 1 to 1581 of , nucleotides 1 to 1389 of SEQ ID NO: 38, nucleotides 1 to 1347 of SEQ ID NO: 40, nucleotides 1 to 2157 of SEQ ID NO: 42 nucleotides, nucleotides 1 to 1395 of SEQ ID NO: 44, nucleotides 1 to 1905 of SEQ ID NO: 46, or nucleotides 1 to 2484 of SEQ ID NO: 48 , where T can be U;

(b) have with SEQ ID NOs:26,28,30,32,34,36,38,40,42,44,46 or the polynucleoside of the sequence described in 48 having at least 80% homology sequence acid;

(c) a polynucleotide encoding a chimeric antigen whose sequence is encoded by DNA contained in one of the following plasmids designated for deposit with the International Depository Authority of Canada (Health Canada The pFastBacHTa HBV S1/S2-TBD, pFastBacHTa HBV core-TBD with the registration numbers 080504-03, 080504-04, 080504-05, 080504-02, and 080504-01 in the Microbiology Office (Bureau of Microbiology at Health Canada) , pFastBacHTa HCV core (1-177)-TBD, pFastBacHTa HCVNS5A-TBD, and pFastBacHTa HCV E2-TBD.

(d) A polynucleotide encoding a chimeric antigen, the antigen sequence being: amino acids 1 to 442 of SEQ ID NO: 27, amino acids 1 to 668 of SEQ ID NO: 29, and amino acids 1 to 668 of SEQ ID NO: 31 Amino acids 1 to 450, amino acids 1 to 431 of SEQ ID NO: 33, amino acids 1 to 598 of SEQ ID NO: 35, amino acids 1 to 527 of SEQ ID NO: 37, amino acids 1 to 527 of SEQ ID NO : 1st to 463rd amino acid of 39, 1st to 449th amino acid of SEQ ID NO: 41, 1st to 719th amino acid of SEQ ID NO: 43, 1st to 465th amino acid of SEQ ID NO: 45, SEQ ID NO: 45th amino acid ID NO: amino acids 1 to 635 of 47, or amino acids 1 to 828 of SEQ ID NO: 49.

(e) a polynucleotide encoding a chimeric antigen-associated protein, which is at least 90% identical to the following entire amino acid sequence, which is: amino acids 1 to 442 of SEQ ID NO: 27, SEQ ID NO: 27 ID NO: amino acids 1 to 668 of 29, amino acids 1 to 450 of SEQ ID NO: 31, amino acids 1 to 431 of SEQ ID NO: 33, amino acids 1 to 598 of SEQ ID NO: 35 , SEQ ID NO: amino acids 1 to 527 of 37, amino acids 1 to 463 of SEQ ID NO: 39, amino acids 1 to 449 of SEQ ID NO: 41, amino acids 1 to 719 of SEQ ID NO: 43 amino acids, amino acids 1 to 465 of SEQ ID NO: 45, amino acids 1 to 635 of SEQ ID NO: 47, or amino acids 1 to 828 of SEQ ID NO: 49.

(f) a polynucleotide that is fully complementary to the polynucleotide of any one of (a)-(d); and

(g) a polynucleotide that selectively hybridizes to the polynucleotide of (a)-(f) under stringent conditions.

The present invention also provides recombinant DNA or RNA molecules comprising chimeric antigen polynucleotides, and analogs or homologues thereof, including but not limited to phage, plasmid, phagemid, cosmid, YAC (yeast artificial chromosome ), BAC (bacterial artificial chromosome), and various viral and non-viral vectors known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, e.g., Sambrook et al., 1989, supra).

The present invention also provides host-vector systems comprising recombinant DNA molecules comprising chimeric antigen polynucleotides in suitable prokaryotic or eukaryotic host cells, and analogs or homologues thereof. Examples of suitable eukaryotic host cells include: yeast cells, plant cells, or animal cells, such as mammalian cells or insect cells (e.g., baculovirus-infected cells, such as Sf9, Sf21, Drosophila S2 or High Five ^™ cells) . Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other infectable or transformable prostate cancer cell lines, primary cells (PrEC), and a number of mammalian cells commonly used for recombinant protein expression (such as COS, CHO, 293, 293T cells). More specifically, the polynucleotide comprising the coding sequence of the chimeric antigen or its fragment, analog or homologue can be used to generate its chimeric antigen using any number of host-vector systems routinely used and well known in the art. antigen.

A wide range of host-vector systems suitable for expression of their chimeric antigens are available, see for example, Sambrook et al., 1989, supra; Ausubel, supra, at pages 1.0.1-1.16.16, 9.01-9.17.3 , and 13.4.1-13.6.5). Preferred vectors for expression in insect cells include, but are not limited to, pFastBac HTa (Invitrogen). Using such expression vectors, chimeric antigens can be expressed in multiple insect cell lines including, for example, Sf9, Sf21, DrosophilaS2, and High Five. Alternatively, preferred yeast expression systems include: Saccharomyces cerevisiae, human yeast probe, Pichia pastoris, and Pichiaaugust. The host-vector system of the invention is used to generate chimeric antigens.

Chimeric antigens or analogs or homologues thereof can be produced by transfecting cells with constructs encoding the chimeric antigens. For example, Sf9 cells can be transfected with an expression plasmid encoding a chimeric antigen, or an analog or homologue thereof, expressed in the Sf9 cells, and the chimeric antigen isolated using standard purification methods. Various other expression systems known in the art can be employed. Expression constructs encoding a leader peptide incorporated in frame with the coding sequence of the chimeric antigen can be used for the generation of a secreted form of the chimeric antigen.

As described herein, the redundancy of the genetic code allows for variations in the gene sequence of the chimeric antigen. In particular, it is known in the art that specific host species usually have specific codon biases, so skilled artisans can modify the disclosed sequences according to the desired host bias. For example, preferred analog codon sequences typically have rare codons (ie, codons that are used less than about 20% of the known sequences of the desired host) replaced by codons of higher frequency. Codon bias for a particular class can be calculated, for example, by using codon usage tables available on the Internet (eg, at the World Wide Web URL www.kazusa.or.jp/codon ).

Additional sequence modifications are known to increase protein expression in the cellular host species. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are detrimental to gene expression. The GC content of the sequence is adjusted to an average level for a particular cellular host, calculated with reference to known genes expressed in the host cell. Where possible, this sequence was modified to avoid the expected hairpin-like secondary mRNA structure. Other useful modifications include the addition of a translation initiation consensus sequence at the beginning of the open reading frame as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Those skilled in the art will appreciate that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5' AUG codon is abolished only in rare cases (see, e.g., Kozak Proc Nat'l Acad Sci USA 92(7):2662-2666(1995) and Kozak Nucl Acids Res 15(20):8125-8148(1987)).

G. Pharmaceutical Compositions of the Invention

One aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a chimeric antigen comprising an immune response domain and a target binding domain, wherein the target binding domain comprises an antibody fragment. In therapeutic applications, the pharmaceutical composition may be administered to a patient in an amount sufficient to elicit effective B cells, cytotoxic T lymphocytes (CTL) and/or helper T lymphocytes (Th) against the antigen. Responding to and preventing infection is sufficient to treat or at least partially inhibit or alleviate symptoms and/or complications of a disease or disorder. Effective amounts for this use will depend upon, for example, the particular composition being administered, the mode of administration, the stage and severity of the disease being treated, the weight and general health of the subject, and the preference of the prescribing physician. judge.

Doses for initial therapeutic immunization (with chimeric antigens) are generally in the unit dose range, with lower values of about 1, 5, 50, 500, or 1,000 ng and higher values of about 10,000 μg; 20,000 μg; 30,000 μg; or 50,000 μg. Dosage values for humans generally range from about 500 ng to about 50,000 μg per 70 kg body weight of the intended subject. The chimeric antigen is administered in increasing doses ranging from about 1.0 ng to about 50,000 μg, depending on the response and disease of the target subject, according to incremental usage over several weeks to months. Administration should continue until at least until clinical symptoms or laboratory tests indicate that the disease has been prevented, suppressed, alleviated or eliminated and also for the period thereafter. Dosage, route of administration, and dosage schedule can be adjusted according to methodology known in the art.

The chimeric antigen in human unit dosage form is typically included in a pharmaceutical composition comprising a human unit dosage of an acceptable carrier (in one embodiment an aqueous carrier) and in a volume/unit known to those skilled in the art. Quantities for administering such polypeptides to humans (see, e.g., Remington: The Science and Practice of Pharmacy, ^20th Edition, A. Gennaro, Editor, Lippincott Williams & Wilkins, Baltimoe, Md., 2000). Various factors can affect the ideal dosage for a particular situation, as will be apparent to those skilled in the art. Such factors include, for example, the half-life of the chimeric antigen, the binding affinity of the chimeric antigen, the immunogenicity of the composition, the desired steady-state concentration level, the route of administration, the frequency of treatment, and the therapeutic methods of the invention. Effects of other substances used in combination, and the health status of a particular target subject.

In some embodiments, compositions of the invention are used in serious disease states, ie, life-threatening or potentially life-threatening conditions. In such cases, due to the relative non-toxicity of the chimeric antigens of the preferred compositions of the invention, administration of chimeric antigens in excess of these stated doses is possible and considered desirable by the treating physician.

The concentration of the chimeric antigens of the invention in the pharmaceutical formulation can vary widely, i.e., from less than about 0.1%, usually at or at least about 2%, up to as much as 20% to 50% or more by weight, and will vary according to The specific application method selected is mainly selected according to the fluid volume, viscosity and the like.

The pharmaceutical composition may be delivered by any route known in the art, such as parenteral, intrathecal, intravascular, intravenous, intramuscular, transdermal, intradermal, subcutaneous, intranasal, topical, oral, rectal, vaginal , lung, intraperitoneal route delivery. Preferably, the composition is delivered parenterally, eg subcutaneously or intradermally.

The pharmaceutical composition can be prepared by mixing the desired chimeric antigen with suitable excipients suitable for the desired route of administration. In the preparation of the pharmaceutical composition of the present invention, the chimeric antigen is usually mixed with excipients, diluted with excipients or encapsulated in a carrier, and the carrier can be in the form of capsules, pouches, paper or other containers. When a pharmaceutically acceptable excipient acts as a diluent, it can be a solid, semi-solid, or liquid substance, which acts as an excipient, carrier, or medium for a therapeutic agent. Thus, the composition may be in the form of: tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) Medium), ointments, soft and hard capsules, suppositories, sterile injectable solutions, and sterile packaged powders containing, eg, up to 10% by weight of the chimeric antigen.

Some examples of suitable excipients include, but are not limited to, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, Crystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulation may additionally include: lubricating agents, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methylparabens and hydroxypropylparabens; sweetening agents; and fragrances. The compositions of the invention can be formulated so as to provide immediate, sustained or delayed release of the chimeric antigen following administration of the chimeric antigen to a subject using methods known in the art. (See, e.g., Remington, supra, at pages 903-92 and pages 1015-1050).

To prepare solid compositions (eg, tablets), the chimeric antigen is mixed with a pharmaceutical excipient to form a solid preformulation composition comprising a homogeneous mixture of chimeric antigens of the invention. When it is said that these preformulation compositions are homogeneous, it means that the chimeric antigen is uniformly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the invention may be coated or otherwise compounded to provide a dosage form which has the advantage of prolonged action. For example, the tablet or pill may comprise an inner dosage and an outer dosage composition, the latter being in the form of a shell of the former. The two components can be separated by an enteric layer that acts to prevent disintegration in the stomach and allows the inner component to enter the duodenum intact or for delayed release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials such as shellac, cetyl alcohol and cellulose acetate.

The novel compositions of the present invention may be incorporated into liquid forms for buccal or parenteral administration, including aqueous solutions, syrups to suit the taste, aqueous or oily suspensions, and edible oils such as corn oil. , cottonseed oil, sesame oil, coconut oil, or peanut oil), as well as elixirs and similar pharmaceutical excipients.

In preparing compositions for parenteral administration, strict attention is paid to the necessity of tonicity adjustment in order to reduce irritation. Reconstitutable compositions are sterile solids packaged in dry form. A reconstitutable composition is preferred because it is more stable when stored as a dry solid than in a liquid ready for immediate application. The dry solids are usually packaged in sterile containers closed with butyl rubber to ensure storage of the solids in the optimum humidity range. Reconstitutable dry solids are formed by dry-fill, spray-drying, or freeze-drying methods. Descriptions of these methods can be found, for example, in Remington, supra, at pages 681-685 and 802-803.

Compositions for parenteral injection are usually diluted and the ingredients present in higher proportions are excipients. The excipient is generally therapeutically inactive and nontoxic, but delivers the chimeric antigen to body tissues in a form suitable for absorption. Absorption is usually most rapid and complete when the chimeric antigen is delivered as an aqueous solution. However, changing or substituting an excipient with a water-miscible liquid for an excipient with a water-immiscible liquid can affect the rate of absorption. Preferably, the maximum excipient for the composition is isotonic saline. In preparing such compositions suitable for injection, one can employ aqueous excipients, water-miscible excipients, and non-aqueous excipients.

Additional substances may be included in the injectable compositions of the invention in order to improve or maintain the quality of the composition. Thus, added substances can affect solubility, provide comfort to the target subject, increase chemical stability, or prevent the growth of microorganisms in the formulation. Thus, the composition may include suitable solubilizers, substances that function as antioxidants, and substances that function as preservatives to prevent the growth of microorganisms. These materials may be present in amounts suitable to their function, but which do not adversely affect the action of the composition. Examples of suitable antimicrobial agents include: thimerosal, benzethonium chloride, benzalkonium chloride, phenol, methylparaben, and propylparaben. Suitable antioxidants can be found in Remington, supra, at p. 1015-1017.

In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like can be used for administration of the chimeric antigens of the invention. In particular, the compositions of the invention may be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles or the like. Alternatively, the compositions of the invention may be covalently or non-covalently attached to the surface of such a delivery vehicle.

Compositions applied via liposomes can also be used to: 1) target chimeric antigens to specific tissues, such as lymphoid tissue; 2) selectively target chimeric antigens to antigen-presenting cells; or 3) improve peptide combination half-life of the substance. Liposomes include: emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellae and the like. In these formulations, the chimeric antigen is delivered as a component of liposomes, alone or in combination with molecules that bind to prevalent receptors in lymphocytes (such as monoclonal antibodies that bind to the CD45 antigen). ) or in combination with other therapeutic or immunogenic components. Thus, liposomes filled or modified with the desired chimeric antigen of the invention can go directly to the site of the lymphocyte where the liposome subsequently delivers the chimeric antigen. Liposomes used according to the invention are formed from standard vesicle-forming lipids, which generally comprise neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid typically takes into account, eg, liposome size, acid instability and stability of the liposomes in the bloodstream. A variety of methods can be used to prepare liposomes, such as Szoka, et al., Ann.Rev.Biophys.Bioeng.9:467-508 (1980) and U.S. Patent No. 4,235,871, No. 4,501,728, No. 4,837,028, No. as described in No. 5,019,369. The liposomal suspension containing the chimeric antigen can be administered intravenously, topically, etc. in a dose that varies depending on the mode of administration, the chimeric antigen being delivered, and the stage of the disease being treated.

Compositions for inhalation or insufflation include pharmaceutically acceptable solutions and suspensions in aqueous or organic solvents or mixtures thereof and powders. The liquid or solid composition may contain suitable pharmaceutically acceptable excipients as described herein. The compositions may be administered by oral or nasal inhalation routes for local or systemic action. Compositions in pharmaceutically acceptable solvents can be nebulized with the aid of inert gases. The nebulized solution can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a mask leg or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from devices that deliver the formulation in a suitable manner.

Another formulation for use in the methods of the invention employs transdermal delivery devices ("patches"). Such transdermal patches can be used to provide continuous or discontinuous infusions of the chimeric antigens of the invention in controlled quantities. The composition and use of transdermal patches for the delivery of pharmaceutical formulations is well known in the art. See, eg, US Patent No. 5,023,252, incorporated herein by reference. Such blocks may be configured for continuous, pulsatile, or on-demand delivery of pharmaceutical formulations.

Furthermore, it may be advantageous to include at least one antiviral therapeutic or chemotherapeutic agent in addition to the chimeric antigen and pharmaceutical excipients. Antiviral drugs include, but are not limited to, peptidomimetics (such as amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir), Polynucleotides (such as ampligen and fomivirsin), purines/pyrimidinones (such as abacavir, acyclovir, adefovir, cidofovir, Cytarabine, didanosine, dideoxy vidarabine, adefovir dipivoxil (dipivoxil), eduridine, emtricitabine, entecavir, famciclovir, ganciclovir, iodine , isoprinosine, lamivudine, MADU, valacyclovir, solivudine, stavudine, tenofovir, trifluridine, valacyclovir, valganciclovir, arabinose adenosine, zalcitidine, and zidovudine), sialic acid analogs (eg, oseltamivir and zanamivir), acetylmorphan, acetylleucine monoethanolamine, amantadine, amidelomycin Adivirine, capravirine, delavirdine, n-docosanol, efavirenz, sodium foscarnet, interferon-α, interferon-β, interferon-γ, Kaitoza, lysozyme, Methylthizone, morpholinidine, nevirapine, pentafuside, pulconaril, podaferenoxine, ribavirin, rimantadine, statamycin, stolonidin, termacamra, and traomantadine. Other suitable antiviral agents are described in Remington: supra, at Chapter 87: Anti-Infectives, pp. 1507-1561, especially pp. 1555-1560. Preferred antiviral therapeutics for inclusion in the pharmaceutical compositions of the present invention include: adefovir, adefovir dipivoxil, entecavir, lamivudine and ribavirin.

In some embodiments it is desirable to include at least one component that primes B-lymphocytes or T-lymphocytes in the pharmaceutical compositions of the invention. Lipids are thought to be substances that can trigger CTLs in vivo. For example, palmitic acid residues can be linked to the ε- and α-amino groups of lysine residues, followed by, for example, one or more linking residues (e.g., Gly, Gly-Gly-, Ser, Ser-Ser , or the like) are linked to the immunogenic peptide. Lipidated peptides can then be applied, either directly within micelles or microparticles, or incorporated into liposomes, or emulsified in an adjuvant such as incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid linked to the ε- and α-amino groups of lysine, which is linked to the immunogenic peptide by a linkage (eg Ser-Ser) the amino terminus.

As another example of a lipid that elicits a CTL response, an E. coli lipoprotein such as tripalmitoyl-S-glycerylcysteinylseryl-serine ( _P3 CSS) is covalently linked to a suitable peptide can be used to elicit virus-specific CTL (see, e.g., Deres, et al., Nature 342:561 (1989)). The chimeric antigen of the present invention can be linked to, e.g., P ₃ CSS, and lipopeptide Administered to an individual to specifically elicit an immune response against an antigen of interest.

Although the compositions of the invention should not require the use of adjuvants, adjuvants can be used. Various adjuvants can be used to enhance the immune response, depending on the host species, and adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels (such as aluminum hydroxide), surface active substances (such as Lysolecithin, detergents, complex polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol) immunostimulatory polynucleotide sequences, and potentially effective human adjuvants (such as BCG ( BCG) and Corynebacterium small). Other adjuvants are also well known in the art.

H. Products

Another aspect of the invention provides an article of manufacture comprising a container containing a composition comprising a chimeric antigen, suitable for injection or reconstituted for injection, in combination with printed labeling instructions providing A description of how to administer the composition parenterally (eg subcutaneously, intramuscularly, intradermally, nasally or intravenously). The composition will be contained in any suitable container which will not significantly interact with the composition and which will be labeled with a suitable label stating that the composition is for parenteral use. The labeling instructions associated with this container will be consistent with the method of treatment described above. The container containing the composition of the present invention may be a container with a liquid composition suitable for injection, with a suitable injection needle and syringe so that the patient, doctor, nurse, or other practitioner can administer the chimeric antigen. Alternatively, the composition may be a dry or concentrated composition comprising a soluble form of the chimeric antigen associated with or diluted with an aqueous or non-aqueous vehicle so that The composition is dissolved or suspended. Alternatively, the container may have a suspension in a liquid or may be the insoluble form of the salt combined with a vehicle in which the insoluble form of the salt can be suspended. Suitable containers are discussed in Remington, supra, pages 788-789, 805, 850-851 and 1005-1014.

The kits of the present invention generally comprise the aforementioned container and one or more of the desired materials from a commercial and user standpoint (including buffers, diluents, filters, injection needles, syringes, and package inserts with instructions for use). ) of other containers. The container may be labeled to indicate that the composition is for a particular therapeutic or non-therapeutic use, and may also indicate directions for in vivo or in vitro use, such as those described above. Instructions for use and/or other information may also be included on an insert within the kit.

V. Examples

The following non-limiting examples provide further illustration of the invention.

A. Example 1: Construction of TBD expression vector

The murine IgGl DNA sequence encoding the amino acids of the _CH1 -hinge- _CH2 - _CH3 region portion was produced from mRNA isolated from a hybridoma (2C12), which produced a mAb directed against the HBV surface antigen (sAg). Total mRNA was isolated using Trizol(R) reagent (Gibco BRL cat. No. 15596-026) and target binding structures were generated by reverse transcription-PCR (RT-PCR) using Superscript First-strand Synthesis (Invitrogen Cat. No. 11904-018) domain (TBD; fragment of a murine immunoglobulin). The PCR primers contained a linker sequence encoding a linker peptide - SRPQGGGS- (SEQ ID NO: 1) at the 5' end, a unique Not I site at the 5' end and a unique Hint III restriction site at the 3' end. The generated cDNA contained (5'Not I)-Linker- _CH1 (VDKKI) (SEQ ID NO: 2).-Hinge- _CH2 - _CH3- (3'Hind III). After digestion with the corresponding enzymes, this fragment was ligated with the pFastBac HTa expression vector plasmid (Invitrogen) using the same restriction sites. The 5' end primer for PCR amplification was (sense) 5' TGTCATTCTGCGGCCGCAAGGCGGCGGATCCGTGGACAAGAAAATTGTGCCCAGG (SEQ ID NO: 3) and the 3' end primer was (antisense) 5' ACGAATCAAGCTTTGCAGCCCAGGAGAGTGGGAGAG (SEQ ID NO: 4), which contained Not I and Hind III sites. A protocol for directional cloning follows. The resulting fragments were digested with the corresponding enzymes, purified on agarose gels and cloned into vector plasmids. The correctness of the DNA sequence and ORF was verified by standard sequencing methods.

After cloning of the DNA encoding the target binding domain into the pFastBac HTa donor plasmid, the recombinant protein was expressed using the Bac-to-Bac ^™ baculovirus expression system (Invitrogen). The cloned gene was transferred into the bacmid by site-specific transposition in E. coli strain DH10Bac. DH10Bac cells contain the shuttle vector, which confers kanamycin resistance, and a helper plasmid, which encodes a transposase and confers tetracycline resistance. A 100 μl aliquot of the appropriate DH10Bac cells was thawed on ice, a pFastBacHTa-based plasmid was added, and the mixture was incubated on ice for 30 minutes. The mixture was heat-shocked at 42°C for 45 seconds, then cooled on ice for 2 minutes. This mixture was then added to 900 μl LB medium and incubated at 37° C. for 4 hours. The transformed cells were serially diluted with LB to 10 ^-1 and 10 ^-2 , and 100 μl of each dilution was placed on an LB agar plate (the agar plate was supplemented with 50 μg/ml kanamycin, 7 μg/ml genta Mycin, 10 μg/ml tetracycline, 100 μg/ml X-gal and 40 μg/ml IPTG) and incubated at 37°C for at least 36 hours. Gentamicin resistance conferred by pFastBac HTa, X-gal, and IPTG (isopropylthio-β-D-galactoside) was used to distinguish white colonies (recombinant plasmid) from blue colonies (non-recombinant plasmid) . Pick out the white colony and inoculate it in 2ml LB medium (the medium is supplemented with 50μg/ml kanamycin, 7μg/ml gentamycin and 10μg/ml tetracycline) and incubate overnight at 37°C with shaking . A small sample of the overnight culture was taken with a sterile loop and streaked onto a fresh LB agar plate (the agar plate was supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamycin, 10 μg/ml tetracycline, 100 μg/ml X-gal and 40 μg/ml IPTG) and incubated at 37°C for at least 36 hours to confirm the white phenotype. Recombinant bacmids were isolated by standard methods (Sambrook, supra), DNA samples were dissolved in 40 μl TE (10 mM Tris-HCL pH 8, 1 mM EDTA) and used for transfection.

To generate baculovirus, bacmids were transfected into Sf9 insect cells. Sf9 cells (9×10 ⁵ ) were seeded in each well of a 6-well cell culture dish (35 mm well) in 2 ml ESF921 (expression system) and were able to attach at 27° C. for at least 1 hour. Transfection was performed using Cellfiction(R) reagent (Invitrogen, Cat. No. 10362-010) according to the protocol provided by the supplier of the Sf9 cells. After transfection, cells were incubated at 27°C for 72 hours. The culture medium containing the baculovirus was collected and stored at 4°C protected from light (in the dark).

Transfection efficiency was confirmed by measuring the production of baculoviral DNA. The isolated baculovirus DNA was subjected to PCR to screen for the inserted gene encoding TBD. Primers used were (sense) 5' TATTCCGGATTATTCATACCG (SEQ ID NO: 5) and 3' (antisense) 5' CTCTACAAATGTGGTATGGC (SEQ ID NO: 6). The amplified product was run on an agarose gel (0.8%). Expression of heterologous proteins in cells was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting probed with a 6xHis-tagged monoclonal antibody (Clontech).

Once baculovirus production and protein expression are confirmed, virus production is amplified to generate a concentrated stock of baculoviruses carrying the gene encoding the binding domain of interest. Amplifying baculoviruses by at least two-fold is standard practice in the art and was adhered to throughout the protocols described herein. Following the second cycle of amplification, the concentration of baculovirus produced was quantified using a plaque assay according to the protocol described by the manufacturer of the kit (Invitrogen). At the same time, the optimal concentration of virus infection of High Five cells and the optimal time point of desired protein production were also established. Typically, MOI 1 and a time period of 48 hours were used for expression of TBD.

B. Example 2: Construction of Chimeric Antigen Expression Vector

DNA encoding the desired viral antigen was generated from the template using the PCR method using the 5' sense and 3' antisense primers in Table 2. The resulting amplified fragments contain a unique restriction site "5' enzyme" at the 5' end and a unique restriction site "3' enzyme" at the 3' end, each of which is used for ligation.

Table 2: Construction of chimeric antigen vectors viral antigen sense primer antisense primer template 5' enzyme 3' enzyme HBV S1/S2 SEQ ID NO: 7 SEQ ID NO: 8 pRSET BHV S1/S2 Bam HI Not I HBV S1/S2/S SEQ ID NO: 9 SEQ ID NO: 10 pAlt HBV 991 Nco I Not I HBV core SEQ ID NO: 11 SEQ ID NO: 12 pAlt HBV 991 Nco I Not I DHBV PreS/S SEQ ID NO: 5 SEQ ID NO: 13 pFastBac Hta PreS/S Eco RI Not I DHBV PreS SEQ ID NO: 5 SEQ ID NO: 14 pFastBac HTa PreS/S Eco RI Not I DHBV Core SEQ ID NO: 15 SEQ ID NO: 16 pRSETB DHBV Core Nco I Not I HCV Core (1-191) SEQ ID NO: 17 SEQ ID NO: 18 pCV-H77c Eco RI Spe I HCV Core (1-177) SEQ ID NO: 17 SEQ ID NO: 19 pCV-H77c Eco RI Spe I HCV NS5A SEQ ID NO: 20 SEQ ID NO: 21 pCV-H77c Eco RI Spe I HCV E1 SEQ ID NO: 22 SEQ ID NO: 23 pCV-H77c Eco RI Spe I HCV E2 SEQ ID NO: 24 SEQ ID NO: 25 pCV-H77c Eco RI Spe I HCV E1/E2 SEQ ID NO: 22 SEQ ID NO: 25 pCV-H77c Eco RI Spe I

The amplified DNA was digested with appropriate 5' and 3' restriction enzymes and ligated into the pFastBacHTa expression vector to generate expression plasmids for viral antigens only. The same DNA fragment was also ligated to the plasmid pFastBac HTa-TBD, as described in Example 1, and subsequently digested with the corresponding enzymes to generate the expression plasmid for the viral antigen fused to the binding domain of interest. The resulting plasmids were used to generate recombinant baculoviruses, as described in Example 1, for subsequent use in the expression of chimeric antigens. The DNA and amino acid sequences of the chimeric antigen are shown in Table 3.

Table 3: Sequences of Chimeric Antigens Construct dna sequence expressed protein HBV S1/S2-TBD SEQ ID NO: 26 SEQ ID NO: 27 HBV S1/S2/S-TBD SEQ ID NO: 28 SEQ ID NO: 29 HBV Core-TBD SEQ ID NO: 30 SEQ ID NO: 31 DHBV PreS-TBD SEQ ID NO: 32 SEQ ID NO: 33 DHBV PreS/S-TBD SEQ ID NO: 34 SEQ ID NO: 35 DHBV Core-TBD SEQ ID NO: 36 SEQ ID NO: 37 HCV Core (1-191)-TBD SEQ ID NO: 38 SEQ ID NO: 39 HCV Core (1-177)-TBD SEQ ID NO: 40 SEQ ID NO: 41 HCV NS5A-TBD SEQ ID NO: 42 SEQ ID NO: 43 HCV E1-TBD SEQ ID NO: 44 SEQ ID NO: 45 HCV E2-TBD SEQ ID NO: 46 SEQ ID NO: 47 HCV E1/E2-TBD SEQ ID NO: 48 SEQ ID NO: 49

C. Example 3: Expression and purification of TBD, viral antigens and chimeric antigens

Normalized multiplicity of infection (MOI) of recombinant bacmids was used to infect High Five ^™ insect cells. For suspension culture, cells were seeded at a density of 3 x ¹⁰⁵ cells/mL and incubated at 27.5 °C with shaking at 138 rpm until the cell density reached 2-3 x ¹⁰⁶ cells/mL. A standard amount of the corresponding recombinant baculovirus was added to the cells. The incubation temperature was 27.5°C, and the appropriate infection period was normalized for individual protein expression. Cells were harvested by centrifugation at 2500 rpm for 10 minutes at 4°C and used for purification of recombinant proteins. Unused fractions of cells were snap frozen in liquid nitrogen and stored at -70°C.

Purify recombinant proteins under denaturing conditions. The cells were incubated in a buffer (lysis buffer) (containing 6M guanidinium-HCl (guanidinium-HCl, guanidinium hydrochloride) in 100 mM sodium dihydrogen phosphate, 10 mM Tris, 300 mM sodium chloride, 10 mM imidazole, pH 8.0) crack. The suspension was sonicated on ice with 5 pulses (1 minute each) at a power setting of 60 W, followed by continuous mixing for 1 hour at room temperature. Centrifuge the lysate at 10,000 x g for 10 min to remove unbroken cells and cell debris. The supernatant was loaded onto a Ni-NTA agarose (Qiagen) bead column (1 x 5 cm/100 mL cell lysate), pre-equilibrated with lysis buffer. After loading, the column was washed with 20 column volumes of 6M guanidinium-HCl (in 100 mM NaH ₂ PO ₄ , 10 mM Tris, 300 mM NaCl, 40 mM imidazole, pH 8.0) (wash buffer 1), followed by 20 column volumes of The column was washed with 8M urea in 100 mM NaH ₂ PO ₄ , 10 mM Tris, 300 mM NaCl, 40 mM imidazole, pH 8.0 (wash buffer 2). The bound protein was eluted with a buffer containing 8M urea, 100 mM NaH ₂ PO ₄ , 10 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8 (elution buffer). Fragments containing the protein were aggregated and dialyzed at 4° C. against various changes of dialysis buffer (10 mM NaH ₂ PO ₄ , 300 mM NaCl). The purified protein was characterized using standard biochemical techniques, including SDS gel electrophoresis, isoelectric focusing, and Western blot analysis using antigens directed against different domains of the expressed protein.

D. Example 4: Breaking tolerance to "self" proteins using chimeric antigen fusion proteins

To assess immune responses to chimeric antigen fusion proteins, mice were immunized with purified HBVS1/S2, TBD, or S1/S2-TBD proteins, and antibody production against each protein was quantified. Proliferation of splenic T cells collected from immunized mice was assessed after stimulation with the respective proteins.

BALB/c mice aged 15 weeks were used for immunization. Mice were injected subcutaneously with S1/S2-TBD (4.15 μg), S1/S2 (4.15 μg) or TBD (4.15 μg) four times at two-week intervals. Blood samples were collected before the start of immunization and one week after each immunization. Sera were prepared from clotted blood samples and used to estimate the levels of antibodies produced by the host animals to the respective injected antigens.

1. ELISA for detecting antibodies against HBV S1/S2, TBD or S1/S2-TBD

A 96-well plate was coated with antigen HBV S1/S2, TBD or S1/S2-TBD at a concentration of 1.0 μg/mL and left overnight at 4°C. The plate was washed with PBS containing 2% BSA. Diluted sera from individual animals were added to each well at different dilution ratios (1:10-1:500) and incubated at 37°C for 1 hour. The plate was washed with PBS (wash buffer) containing 0.05% Tween 20. Dilutions of goat anti-mouse IgG Fab-horseradish peroxidase (HRP) (1 :5000) were added to the wells and incubated at 37°C for 1 hour. The plates were washed with wash buffer and developed with 2-2'azino-bis(3-ethylbenzylthiazoline-6-sulfonate) (KPL, Guildford, UK). The optical density of the generated color in the samples was measured at a wavelength of 405 nm using an ELISA plate reader (Molecular Devices, USA). The negative control used for this experiment was pre-immune serum from the same animal, which was subtracted from all experimental values. The results of mice being immunized with HBV S1/S2-TBD are shown in Table 4. The chimeric antigen elicited a strong antibody response against the chimeric antigen (S1/S2-TBD).

Table 4: Humoral responses to S1/S2-TBD antibody binding level HBV S1/S2-TBD HBV S1/S2 TBD immunized mice HBV S1/S2-TBD 0.192 0.059 0.048 HBV S1/S2 0.073 0.015 0.025 TBD 0.076 0.017 0.036

The antibody responses described herein are multivalent (or multiepitopic) in nature. The results shown in Table 4 indicate that the antibodies produced by immunizing mice with HBV S1/S2-TBD bind to the chimeric antigen and S1/S2 protein targets coated on the plate. Thus, antibodies were raised against the S1/S2 component of the chimeric antigen. Likewise, antibodies produced by HBV S1/S2-TBD immunized mice bound to the target binding domain proteins (Table 4). A chimeric antigen comprising a protein of murine origin can generate a humoral immune response in mice, suggesting that the chimeric antigen can convert a "self" antigen into a "foreign" antigen. Thus, tolerance to proteins treated as "self" proteins can be broken.

2. T cell proliferation assay

Animals were sacrificed one week after the fourth immunization, spleens were removed, and individual cell suspensions were generated. Cells were seeded in triplicate in 96-well plates at a cell density of 4 × ¹⁰⁵ cells/well. They were loaded with 0.1 μg/mL HBV S1/S2, 1.0 μg/mL TBD or 10 μg/mL S1/S2-TBD antigen, respectively. Negative control cells received medium only and positive control for T cell proliferation was 1.0-5.0 μg/mL phytohemagglutinin (PHA). The cell culture was incubated at 37 °C for four days in an atmosphere of 7% _CO2 . Cells in each well were pulsed with 1.0 mCi of ³ [H]-thymidine and incubated for an additional 18 hours, harvested using a TOMTEC MACH3 cell harvester (Hamden, CT, USA) and liquid scintillation using a Wallac Trilux 1450 Microbeta Radioactivity bound to glass fiber filters (Wallac Oy, Turku, Finland) was quantified with a luminescence counter (Wallac, USA). The results are shown in Table 5.

Table 5: Cellular responses to HBV S1/S2-TBD Counts per minute (CPM) 0.1μg/mL 1μg/mL 10μg/mL HBV S1/S2-TBD 36.7±17.0 32.7±5.0 21.3±7.0 HBV S1/S2 10.7±2.1 26.7±9.6 25.7±10.3 TBD 32.7±19.1 17.0±2.6 35.7±27.2 individual cells 21.3±12.5 1.0mg/ml PHA 39.3±18.6

T cell proliferation was observed when stimulated with HBV S1/S2-TBD, S1/S2 or TBD. Immunization with chimeric antigens results in multivalent T cell responses, ie, responses directed against different parts of the same protein. Chimeric antigens comprising murine proteins can generate cellular immune responses in mice, suggesting that the chimeric antigens can convert "self" antigens into "foreign" antigens. Thus, tolerance to proteins treated as "self" proteins can be broken.

E. Example 5: Antigen presentation experiment

The ability of HBV S1/S2-TBD to elicit an immune response was measured using an in vitro antigen presentation assay. Generation of effective T cell responses following multiple stimulations of naive T cells with antigen-loaded antigen-presenting cells (APCs) such as dendritic cells (DCs) is achieved by enumerating (quantifying) the increase in the number of antigen-specific T cells and the generation of T cells The capacity of Th1 cytokine IFN-γ was estimated.

1. Selection of Monocytes by Adhesion

Peripheral blood mononuclear cells (PBMC) were thawed by adding AIM-V (at a ratio of 9ml AIM-V to 1ml frozen cells). The cells were then centrifuged at 200 x g for 5 minutes, the supernatant was removed, and the cells were resuspended in AIM-V/1% matched serum and added to 100 ml dishes or T-25 in a culture bottle. Incubate PBMCs in a humidified _incubator at 37 °C with 7% CO for 1 h. To remove non-adherent cells, the culture was triturated several times, the supernatant was discarded, and the cells were washed once with AIM-V medium. Mononuclear cells were collected with a cell scraper and centrifuged at 300 x g for 5 min. Cell pellets were resuspended at 2 x ¹⁰⁶ cells/ml in AIM-V/2.5% matched serum and seeded in 24-well dishes. Cytokines IL-4 and GM-CSF (1000 IU/ml each) were added to induce differentiation of monocytes into immature DCs.

2. Fast or slow antigen presentation experiments

For rapid antigen presentation assays (APA), antigen was added to immature DCs and isolated within 4-24 hours. After an additional 24 hours, antigen-loaded immature monocytes were induced to mature monocytes by incubation with PGE2 (1 μM), IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 24 hours. Mature DCs are then incubated with autologous T cells (primary stimulation). T cells were generated from the same PBMC as DCs by negative selection using the Magnetic T Cell Isolation Kit (Dynal) according to the manufacturer's instructions.

T cells were subsequently restimulated with antigen-loaded mature DCs for 7 days in the presence of IL-2 (20 IU/ml), IL-7 (10 ng/ml) and IL-15 (5 ng/ml). After 7 days of incubation, T cells were restimulated a third time with antigen-loaded mature DCs. The third stimulation lasted 6 hours, after which T cells were harvested and immunostained for CD3, CD3 and IFN-γ expression, analyzed by flow cytometry.

For slow APA, monocytes were allowed to differentiate into immature DCs in the presence of GM-CSF and IL-4 for 5 to 6 days before antigen addition. Two hours after antigen addition, immature DCs were matured by TNF-α (10 ng/ml) and INF-α (50 IU/ml). Seven days after isolation, mature DCs were co-cultured (prime stimulation) with autologous T cells (as described above).

Subsequently, T cells were restimulated with antigen-loaded mature DCs for 7 days in the presence of IL-2, IL-7, and IL-15. After 7 days of incubation, the cells were restimulated a third time with antigen-loaded mature DCs. After 18 hours of incubation, the T cells were harvested and immunostained for CD3, CD8 and IFN-γ expression and analyzed by flow cytometry.

3. BMC antigen presentation experiment

In this experiment, the initial culture included total PBMCs (i.e., lymphocytes and monocytes), which were incubated with antigen and IL-2, and it was assumed that this system resembles an in vivo immune response because all cell types are present and involved (Maini, MK et. al J. Exp. Med. 191:1269-1280, 2000). PBMCs are thawed, washed and rapidly incubated with antigen. After four days of incubation to allow antigen uptake and presentation, IL-2 (20 IU/ml) was added and left for an additional 8 days (ie day 12 of the experiment). Two days before the second stimulation (ie day 10 of the experiment), DCs were isolated by adhesion as described above and rapidly incubated with GM-CSF, IL-4 and antigen for 24 hours. For fast API, immature DC were allowed to differentiate for 24 hours after addition of _PGE2 , IL-1β and TCF-α. Loaded mature DCs were subsequently added to PBMC cultures in the presence of IL-2, IL-7 and IL-15 (second stimulation, day 12 of the experiment). The third stimulation occurred on day 21 of the experiment with antigen-loaded mature DC prepared two days before day 21 of the experiment. After 6 hours incubation, the T cells were harvested and immunostained for CD3, CD8 and IFN-γ expression and analyzed by flow cytometry.

For all antigen presentation experiments described above, at the end of the experiment, some T cells were incubated for an additional 3 to 5 days and specific T cells were checked by tetramer analysis (see below).

4. HBV S1/S2 elicits T cell responses against HBV S1 and S2 peptides

PBMC APA was used to generate T cells, which were subsequently assessed for their antigen specificity. Therefore, PBMCs from healthy HLA-A2 individuals were cultured at 5 x ¹⁰⁵ cells/ml in 96-well plates in AIM-V containing 2.5% matched serum. Antigen (ie 10 μg/ml S1/S2-TBD) was added and cells were incubated at 37°C for 4 days. IL-2 was then added at 20 IU/ml and the cells were cultured for an additional 8 days with medium changes (AIM-V/2.5% corresponding serum and 20 IU/ml IL-2) every 2-3 days. Most of the cells retained at the end of the 12-day culture were T cells, and in the presence of IL-2 (20IU/ml), IL-7 (10ng/ml), and IL-15 (5ng/ml) with autologous These T cells were re-stimulated with the source of antigen-loaded mature DC.

Antigen-loaded mature DCs for the second and third stimulation of T cells in APA were generated 48 hours later using the method described below. Monocytes were isolated from bulk PBMCs by adhesion to plastic tissue culture dishes. The cells (approximately 85% of which were monocytes (CD11c+, CD14+, CD19-, and CD3-) as determined by FACS) were cultured at 1×10 ⁵ cells/well in a 96-well plate containing 1000 IU/ml 100 μl of AIM-V/2.5% matched serum for cytokines IL-4 and GM-CSF, 4 hours later antigens such as S1/S2-TBD were added. After 20 hours of incubation, the resulting immature DCs were differentiated by culturing for an additional 24 hours in the presence of PGE2 (1×10 ^-6 M), IL-1β (10ng/ml) and TNF-α (10ng/ml) For mature DC.

T cells were cultured for 7 days after the second stimulation with medium changes (AIM V with 2.5% matched serum and 20 IU/ml IL-2) every 1-2 days. T cells (cultured for 19 days) were then stimulated a third time with antigen-loaded mature DCs (generated by the 2-day process described above) in the presence of IL-2, IL-7, and IL-15 (as described above), And the production of IFN-γ was assessed after 6 hours of culture or cultured for 5 days (replace the medium with AIMV/2.5% corresponding serum and 20IU/ml IL-2 every 1-2 days), and then assessed by HBVpreS tetramer Specificity of T cells to HBV preS antigen (day 24 in culture).

Tetramer analysis was performed with customally synthesized iTag MHC class I tetramers (Beckman Coulter) according to the manufacturer's protocol. Cells were therefore harvested, washed and transferred to 96-well v-bottom plates at ~2 x ¹⁰⁵ cells/well, 20 μl. HLA-A ^* 0201preS1 tetramer (GMLTPVSTI, SEQ ID NO:50) or preS2 conjugated with mAbs specific for CD3 (anti-CD3-FITC) and CD8 (anti-CD8-cysteine-chromium) and 2 μl of PE Tetramer (NIASHISSI, SEQ ID NO: 51 ) together labeled the cells at 20°C for 30 minutes. The cells were then washed, fixed with 2% paraformaldehyde in PBS and transferred to 5 ml FACS tubes. 80,000-100,000 cells per sample were acquired on a FACSCalibur flow cytometer (BD Bioscience). Analysis was performed using CellQuest software (BD Biosciences), which gates on the active (FSC/SSC based) CD3+ population, and the percentage of CD8+ cells labeled with tetramers was determined. When PBMC were cultured with 10 μg/ml of HBV S1/S2-TBD and restimulated twice with HBVS1/S2-TBD-loaded mature DC, the indicated percentage of cells were tetramerized with S1 (Figure 2) and with S2 Objects (Figure 3) were marked as positive. This is in contrast to T cells cultured with unantigen-loaded mature DC, where the number of tetramer positive cells was not significant. Thus, mature DCs loaded with S1/S2-TBD were able to induce the generation of significant numbers of T cells specific for determinants of HBV S1 and HBV S2 antigens.

F. Example 6: Breaking tolerance to DHBV and DHBV antigens using chimeric antigen fusion proteins

DHBV is used as an effective animal model in the development of antiviral therapy for HBV. Pekin ducks congenitally infected with DHBV have been used to study the mechanism of virus replication and to screen antiviral components. Two duck models were used in the present invention. The first was ducks congenitally infected with DHBV. This is similar to the vertical transmission of human HBV-infected persons. The second is a persistent infection model in which newly hatched ducklings are infected with DHBV and these ducklings carry the infection. The second model is similar to horizontal transmission of human HBV infection.

1. Ducks congenitally infected with DHBV

Ducks congenitally infected with DHBV were divided into 2 groups at four weeks of age. Blood samples (1.0 mL) were collected as a control for preimmune antibody levels and weekly prior to vaccination. The test group received 19.95mg/dose subcutaneous injection of DHBV core-TBD chimeric antigen fusion protein on the same day every week until the fifth week. At week six, the dose was doubled and injections were given every four weeks until vaccination was discontinued at week 26. The control group (placebo group) received the same amount of buffer (20 mM sodium phosphate pH 8.0, 300 mM NaCl).

96-well plates were coated with antigen, DHBV core, TBD or DHBV core-TBD at a concentration of 1.0 μg/mL and left overnight at 4°C. The plate was washed with phosphate buffered saline (PBS) containing 2% BSA. Different dilutions (1:10-1:500) of serum from each animal were added to each well and incubated at 37°C for 1 hour. The plate was washed with PBS (wash buffer) containing 0.05% Tween 20. Dilutions of goat anti-duck IgG-HRP (1 :5000) were added to the wells and incubated at 37°C for 1 hour. The plate was washed with wash buffer and developed with 2-2'azino-bis(3-ethylbenzylthiazoline-6-sulfonate) (KPL, Guildford, UK). The optical density of the color produced in the samples was measured using an ELISA plate reader (Molecular Devices, USA). Antibody titers were calculated relative to pre-immune sera from the same animals.

The levels of anti-core antibodies in the sera of ducks congenitally infected with DHBV in ducks from the control and experimental groups at 0, 3, and 6 weeks are shown in Table 6. Although the duck had a chronic DHBV infection, the antibody levels were low due to the chronic nature of the infection and the inability of the immune system to recognize the antigen as a foreign molecule. After immunization with the DHBV core-TBD chimeric antigen, the host's immune system recognizes the viral antigen and generates a humoral immune response against the core antigen already present in the host, thereby breaking the host's tolerance to the viral antigen.

Table 6: Humoral immune response against DHBV core-TBD Antibody binding (OD405) Anti-DHBV core anti-TBD vaccinated duck week 0 0.058±0.005 0.005±0.003 week 3 0.131±0.029 0.092±0.059 week 6 0.166±0.021 0.147±0.038 control group week 0 0.062±0.016 0.003±0.002 week 3 0.074±0.015 0.010±0.005 week 6 0.087±0.012 0.035±0.017

Likewise, the duck's immune system recognizes the TBD component of the chimeric antigen as foreign and mounts an immune response against this portion of the fusion protein as well. Plates were plated with TBD and sera from individual ducks for assessment of antibody levels by ELISA. The results of this study are shown in Table 6.

2. Ducks infected with DHBV after hatching

Normal ducklings were infected with duck serum containing DHBV one day after hatching. This is standard practice in the DHBV research field. The presence of persistent viremia was confirmed using established techniques 4 weeks before the start of immunization. Ducks infected with DHBV were divided into two groups. Blood samples (1.0 mL) were collected from each duck as a control for pre-immune antibody levels, and blood samples were collected weekly prior to vaccination. The experimental group received 19.95 μg/dose subcutaneous injection of DHBV core-TBD fusion protein on the same day every week until the fifth week. At week six, the dose was doubled and injections were given every four weeks until vaccination was discontinued at week 30. Blood samples were collected from a control group that received an equal amount of buffer (20 mM sodium phosphate pH 8.0, 300 mM NaCl).

Antibody levels have been tested in sera collected from ducks at 0, 3, and 6 weeks. The levels of anti-core antibodies in the sera of DHBV-infected ducks after incubation in ducks of the control and test groups are shown in Table 7. Although DHBV has established a persistent infection, antibody levels are low because the immune system cannot recognize the viral antigens as foreign molecules. After immunization with the DHBV core-TBD chimeric antigen, the host's immune system recognizes the viral antigen and generates a humoral immune response against the core antigen already present in the host, thereby breaking the host's tolerance to the viral antigen. Antibody levels against TBD were also elevated (Table 7). Thus, there are multivalent (or multi-epitopic) immune responses directed against different parts of the same chimeric antigen.

Table 7: Humoral immune response against DHBV core-TBD Antibodies bind to: DHBV Core TBD vaccinated duck week 0 0.006±0.011 0.003±0.002 week 3 0.145±0.014 0.072±0.043 week 6 0.170±0.009 0.163±0.052 control group week 0 0.083±0.016 0.008±0.010 week 3 0.112±0.042 0.011±0.007 week 6 0.138±0.041 0.026±0.013

G. Example 7: Chimeric cross-linked HBV sAg-Fc (mouse)

A solution of 100 μg sAg (US Biologicals; Cat#H1910-27) and 100 μg mouse polyclonal IgG Fc fragment (HarlanSera-Lab Ltd., Cat#PP-19-01) was dialyzed against 100mM HEPES (pH8.7) at 4°C. night. The protein solutions were mixed together, and dimethylsuberimide (DMS; Pierce Cat#20700) was immediately added to a final concentration of 10 mM, and the mixture was incubated for 1 hour at room temperature. The reaction was stopped by adding 0.1M Tris HCl pH 7.8. The reaction mixture was loaded on a Sephadex G75 column (0.7 x 12 cm), and the fragments were eluted with phosphate buffered saline. 0.5 ml of fragments were collected and the fragments containing sAg/Fc in a molar ratio of 1:1 (estimated by ELISA using the corresponding antibodies) were pooled.

Pooled fragments were used for antigen presentation experiments (Berlyn, et al., Clin. Immunol. 101:276-283, (2001)). Immature dendritic cells were cultured with GM-CSF/IL4 for 4 days, incubated with sAg-Fc conjugates, and matured in the presence of TNFα and interferon-α. Autologous CD3 ⁺ T cells were added to mature dendritic cells. T cell stimulators were quantified by measuring intracellular interferon-γ production by flow cytometry following three cycles of exposure to mature dendritic cells. The level of intracellular interferon-γ produced in T cells in the presence of the conjugate was much higher than in the presence of only sAg or Fc fragment (Table 8).

Table 8: T cell responses to HbsAg-Fc DMS conjugates % IFN-γ positive T cells no antigen 0.19 Mouse Fc (2.5μg/mL) 0.46 HBsAg (2.5μg/mL) 0.04 HBsAg(2.5μg/mL)+mAb(2C12)(2.5μg/mL) 0.12 HBsAg-Fc DMS conjugate (5.0μg/mL) 0.74

H. Example 8: Antigen presentation experiment

Antigen presentation experiments were performed using human PBMC-derived dendritic cells according to established protocols (Berlyn, et al., supra (2001)). A summary of the protocol used for T cell stimulation experiments is shown below in schematic form.

1. Preparation of Mature Loaded Dendritic Cells

Monocytes were generated from leukocyte-depleted samples of healthy donors and lymphocytes and granulocytes were depleted by incubation with anti-CD2, CD7, CD16, CD19 and CD56 antibodies. This was then incubated with magnetic bead-bound anti-mouse IgG and separated on a magnet (Dynal). Negatively selected cells were >95% pure monocytes by flow cytometry utilizing a broad panel of CD markers (CD14 ⁺ , CD11c ⁺ , ^CD19- , CD3-, ^CD4- , ^{CD64 +} , CD32 ⁺ , CD86 ⁺ , CD16 ^- ) characterizes it. Subsequently, monocytes were incubated with GM-CSF (R&D Systems) in AIMV plus 2.5% corresponding human serum for 4 days to generate immature dendritic cells. Again, an aliquot of the cells was stained by an extensive CD marker panel to ensure the purity and identity of the cells. The cells were then loaded with HBV S1/S2-TBD (5.0 μg/ml), HBV S1/S2 (2.5 μg/ml), or TBD (2.5 μg/ml) for 2-4 hours at 37°C, followed by interferon -α and TNF-α matured the cells for 3 days. Dendritic cells were checked again using flow cytometry (batch of CD markers) to ensure that the cells had undergone proper maturation. The resulting mature loaded dendritic cells were used for T cell stimulation experiments.

2. T cell stimulation experiment: cytokine analysis

T cells were generated from the same PMBC samples as dendritic cells by negative selection using the Magnetic T Cell Isolation Kit (Dynal) according to the manufacturer's instructions. Mature loaded dendritic cells (DC-1) were rinsed thoroughly and added to T cells (day 0). The T cells were incubated with dendritic cells for 7 days. On day 7, the T cells were restimulated with mature, loaded dendritic cells (DC-2). An aliquot of the cells was taken after 2 hours. An aliquot of this cell was incubated with brefeldin A (GolgiPlug ^™ , R&D Systems) for 18 hours and then used in the intracellular cytokine staining experiment described below.

The remaining cells were incubated for an additional 7 days. On day 14, the remaining cells were stimulated with a third batch of mature, loaded dendritic cells (DC-3). An aliquot of the cells was taken after 2 hours. An aliquot of this cell was incubated with brefeldin A (GolgiPlug ^™ , R&D Systems) for 18 hours and then used in the intracellular cytokine staining experiment described below.

For intracellular cytokine staining, cells were stained with anti-CD3-FITC and anti-CD8-cysteine-chromium for 30 min, washed, fixed, permeabilized, and then stained with anti-IFN-γ-PE on ice 30 minutes. Cells were washed and analyzed by flow cytometry (FACScan, BD Biosciences). The results are shown in Table 9.

Table 9: CD3 ⁺ /IFN-γ ⁺ T cells day 21 HBV S1/S2-TBD 6.2±4.6 HBV S1/S2 1.9±1.7 TBD 1.6±0.9 no antigen 0.58±0.21

After removal of aliquots on day 14, the remaining T cells were incubated for another three days and the supernatant was then used to measure the level of secreted interferon-γ by ELISA (Opt E1A ELISA kit, BD Biosciences). T cell stimulation was assessed by measuring the levels of intracellular and secreted interferon-γ. The results are shown in Table 10. At the same concentration, the chimeric antigen S1/S2-TBD induced a higher level of interferon-γ production than the immune response domain or TBD domain of the molecule when tested with the molecule's immune response domain or TBD domain alone Levels of induced interferon-γ production. It should be noted that a 5 μg dose of S1/S2-TBD contains approximately 2.5 μg of each of these components.

Table 10: Levels of Intracellular and Secreted Interferon-γ % IFN-γ positive T cells Secreted interferon-γ (pg/ml) HBV S1/S2-TBD 3.5 60 HBV S1/S2 2.1 18.9 TBD 2.5 11.9 no antigen 0.77 4.4 isolated T cells 0.21 1.6

Various concentrations of S1/S2-TBD were tested for T cell responses. The effect of S1/S2-TBD was greater than that of tetanus toxoid treatment at similar concentrations. At concentrations below 5 μg/mL, the chimeric antigen elicited a concentration-dependent (ie, positively correlated with S1/S2-TBD antigen concentration) increase in interferon-γ production and secretion. After the S1/S2-TBD antigen was presented by dendritic cells, the production and secretion of interferon-γ by CD3 ⁺ T cells increased in a concentration-dependent manner, as shown in Table 11. A low concentration of positive responses would be advantageous relative to the dose necessary for vaccination and the cost of manufacturing the vaccine.

Table 11: Concentration-dependent responses to chimeric antigens % IFN-γ positive T cells Secreted IFN-γ (pg/ml) HBV S1/S2-TBD (1.25μg/ml) 1.5 18 HBV S1/S2-TBD (2.5μg/ml) 4.3 40 HBV S1/S2-TBD (5μg/ml) 3.5 60 HBV S1/S2-TBD (10μg/ml) 4.3 20 Tetanus toxoid 3.3 33 no antigen 0.77 4.4 isolated T cells 0.21 1.6

I. Example 9: Binding and Uptake of Chimeric Antigens

1. Preparation of Mature Loaded Chimeric Antigen

Peripheral blood mononuclear cells (PBMC) were obtained from Ficoll/Histopaque (Sigma) treatment of leukapheresis cell preparations (Berlyn, et al, supra (2001)). Monocytes were isolated from PBMC populations by negative selection using the Monocyte Isolation Kit (Dynal) following the manufacturer's instructions. The purity of monocytes estimated by antibody analysis and flow cytometry (CD14 ⁺ , CD11c ⁺ , CD19 ⁻ , CD3 ⁻ , CD4 ⁻ , CD64 ⁺ , CD32 ⁺ , CD86 ⁺ , CD16 ⁻ ) was greater than 95%. Monocytes were washed twice with AIM-V (Invitrogen) medium containing L-glucose with 1% donor-matched serum (isolated as described in Berlyn, et al., supra (2001)) Aminoamide, streptomycin sulfate (50 μg/mL) and gentamicin sulfate (10 μg/mL). Subsequently, the monocytes were cultured in AIM-V medium containing 2.5% donor-matched serum and the cytokines GM-CSF and IL-4 to differentiate the cells towards the dendritic cell (DC) lineage. Incubate the cells in 12-well tissue culture plates at 37 °C in a 7% _CO2 atmosphere.

Dendritic cells of monocyte origin were collected on days 1 to 4. The cells were then washed once with AIM-V medium with 0.1% BSC (Sigma) and twice with Dulbecco's phosphate buffered saline (Invitrogen) with 0.1% (w/v) BSA (PBSB). Dendritic cells of monocyte origin were used in labeling or binding experiments at 4°C or in binding/uptake experiments at 37°C.

2. Binding of Chimeric Antigens to Mature Dendritic Cells

The extent of S1/S2-TBD binding to mature dendritic cells was compared relative to murine IgG1 and IgG2a. On each day of in vitro culture (from day 0 to day 4) dendritic cells were isolated and treated with S1/S2-TBD (10 μg/mL) or with mouse IgG1 (2C12, the parental mAb that produces TBD) or with IgG2a (G155-178, 90 μg/Ml) was treated at 4°C for 1 hour. The cells were treated with F(ab') ₂ goat anti-mouse Alexa-488 (10 μg/mL) in PBSB for 20 minutes. The cells were rinsed twice with PBSB, resuspended in PBSB with 2% paraformaldehyde (PF), and acquired by a Becton Dickinsion (BD) FACScan configured with CellQuest acquisition and analysis software (BD). Gates were formed on variable cell populations determined by FSC and SSC scatter profiles and > 10,000 events were acquired. To determine the percentage of positive cells, gates were set based on negative control treated cells (labeled isotype control or cells labeled only with F(ab') ₂ goat anti-mouse Alexa-488 (10 μg/mL)). The percentage of specific positive cells was calculated as follows:

(Percentage of positive cells in test sample-percentage of positive cells in control)/(100-percentage of positive cells in control)×100

The relative mean fluorescence intensity (MFI) was determined by subtracting the MFI of the control samples from the MFI of the test samples.

Binding of S1/S2-TBD relative to IgG1 and IgG2a to DC after 1 to 4 days of culture is shown in Table 12.

Table 12: Binding of Chimeric Antigens or Antibodies to Mature Dendritic Cells percentage of specific positive dendritic cells Day 1 day 2 3rd day day 4 HBV S1/S2-TBD 91.9 98.5 88.3 97.9 IgG1 21.2 19.5 29.3 49.1 IgG2a 28.0 17.4 14.3 13.5

S1/S2-TBD binding was significantly greater than IgG1 or IgG2a binding, and S1/S2-TBD binding was more pronounced on day 1 than on day 4. These experiments demonstrate that S1/S2-TBD is efficiently bound to mature dendritic cells.

3. Uptake of Chimeric Antigen by Mature Dendritic Cells

To determine the extent of uptake of a chimeric antigen (e.g., HBV S1/S2-TBD) relative to IgG1 and IgG2a, cells were treated with various concentrations of the antigen, IgG1 (2C12, the parental mAb from which the TBD was generated) or IgG2a (G155 -178) were incubated in AIM V medium with 0.1% BSA for 1 hour at 37°C. Cells were washed twice in PBSB and fixed overnight at 4°C in PBS with 2% PF. Subsequently, the cells were washed twice in PBSB and permeabilized with PBS containing 0.1% (w/v) saponin (Sigma) at 20°C for 40 minutes.

The cells were washed twice with PBSB and incubated with F(ab') ₂ goat anti-mouse Alexa-488 (10 μg/mL) in PBSB with 0.1% (w/v) saponin for 20 minutes at 4°C. After washing twice in PBSB, the cells were suspended in PBSB. An alternative to this experiment involved treating cells with chimeric antigen, IgGl, or IgG2a for 10 minutes as described above, followed by the addition of F(ab') ₂ goat anti-mouse Alexa-488 (10 μg/mL) for 50 minutes. The cells were then washed and suspended in PBS with 2% PF.

Cells can be acquired by a Becton Dickinson (BD) FACScan configured with Cellquest acquisition and analysis software (BD). Gates were gated on variable cell populations determined by FSC and SSC scatter profiles and > 10,000 events were acquired. To determine the percentage of positive cells, gates were set based on negative control treated cells (labeled isotype control or cells labeled only with F(ab') ₂ goat anti-mouse Alexa-488 (10 μg/mL)). The percentage of specific positive cells was calculated as follows:

(Percentage of positive cells in test sample-percentage of positive cells in control)/(100-percentage of positive cells in control)×100

The relative mean fluorescence intensity (MFI) was determined by subtracting the MFI of the control samples from the MFI of the test samples.

Uptake of S1/S2-TBD relative to murine and IgG2a was assessed as a function of concentration at day 4 of dendritic cell maturation. This uptake was quantified within 1 hour at 37° C. and the results are shown in FIG. 4 . The uptake (amount) of S1/S2-TBD increases linearly with concentration. IgG1 uptake levels were much lower, whereas IgG2a was only minimally uptaked. Thus, the chimeric antigen S1/S2-TBD was more efficiently taken up by dendritic cells than immunoglobulins.

J. Example 10: Expression of Fc-γ receptors and CD206 on mature DCs

There are many receptors on antigen presenting cells that bind and take up antigens. Fluorescently labeled receptor-specific antibodies were used to assess the abundance of receptors on mature dendritic cells. FACS analysis was used to estimate the percentage of specific receptor positive cells in the total population of dendritic cells. The extent of receptor expression was assessed by determining the relative mean fluorescence density and as a function of relative fluorescence density (Table 13).

Table 13: Expression of antigen-binding receptors on mature dendritic cells Specific positive cells % Relative MFI day 0 Day 1 day 2 3rd day day 4 day 0 Day 1 day 2 3rd day day 4 CD16 20.8 26.3 6.2 0.8 5.9 4.3 10.8 2.7 0.0 1.6 CD32 99.3 97.4 78.9 41.8 37.8 163.4 187.1 70.5 18.0 14.0 CD64 84.0 71.9 18.2 9.6 5.3 28.0 12.8 3.7 2.7 1.0 CD206 45.8 82.5 98.3 99.1 99.3 8.5 373.1 1180.6 1317.3 1680.4

Expression of CD64 (Fcγ receptor 1) in culture decreased over time and was almost negligible at day 4. In contrast, CD32 (Fcγ receptor II) and to a lesser extent CD16 (Fcγ receptor III) continued to be expressed after 4 days of DC culture. On day 0 of culture, there was essentially no expression of CD206 (mannose macrophage receptor). However, it was induced when cultured with IL-4 and GM-CSF, and CD206 was expressed at very high levels on day 4. Thus on day 4, when the antigen was loaded in the antigen presentation assay, the dendritic cells had at least two potential receptors for binding the chimeric antigens CD32 and CD206. Furthermore, they are fully complementary to co-stimulatory molecules (data not shown). Expression of HLA-DR (class II) and HLA-ABC (class I) also increased over time in culture. The co-stimulatory molecules CD86 (B7.2) and CD80 (B7.1) were also expressed throughout the experiment. These results suggest that monocyte-derived dendritic cells differentiate toward mature dendritic cells and are capable of processing and presenting antigens for T cells.

K. Example 11: Correlation of CD32/CD206 Expression and S1/S2-TBD Binding to Mature DCs

There is a direct correlation between CD32/CD206 receptor expression and S1/S2-TBD binding to mature dendritic cells. Since murine IgGl is known to bind human CD32, it was expected that S1/S2-TBD comprising the Fc component of murine IgGl would also bind CD32. Furthermore, S1/S2-TBD, by virtue of its high mannose glycosylation, would also be expected to bind to dendritic cells through the CD206 receptor.

The dot plots in Figure 5 show S1/S2-TBD binding (10 μg/mL) and expression of CD32 and S1/S2-TBD binding and expression of CD206. There is a direct correlation between the degree of S1/S2-TBD binding and the degree of expression of CD32 (which is relatively heterogeneous, ie has a broad range of expression). These results indicate that S1/S2-TBD binds to CD32, and that the higher the expression of CD32, the higher the degree of binding of the chimeric antigen S1/S2-TBD. A dot plot of S1/S2-TBD binding versus CD206 expression indicates that the vast majority of cells expressing CD206 also bind S1/S2-TBD. A small percentage of this cell population is negative for CD206 and thus negative for S1/S2-TBD binding. Thus, both CD32 and CD206 receptors are associated with S1/S2-TBD binding.

L. Example 12: Binding and uptake of S1/S2-TBD mainly via CD32, less via CD206

Uptake of S1/S2-TBD compared to murine IgG1 and IgG2a was assessed as a function of concentration at day 4 of DC maturation. This uptake was quantified over 1 hour at 37°C in the presence of culture medium, mannan (2 mg/ml, Sigma), and/or murine Fcγ (2 mg/ml, Jackson Immunological Research Laboratories). Mannan is a competitive inhibitor of CD206 binding and thus antigen uptake via CD206 on dendritic cells. Fcγ is a competitive inhibitor of CD32 binding, thus CD32 mediates antigen uptake. The results are shown in Table 14.

Table 14: Inhibition of Chimeric Antigen Binding by Fc or Mannan Relative MFI Mannan mouse Fcγ Mannan & Fcγ culture medium 0.5 μg/ml HBVS1/S2-TBD 7.6 0.5 0.6 3.0 2.5 μg/ml HBVS1/S2-TBD 21.5 2.0 3.3 22.6 6 μg/ml HBVS1/S2-TBD 41.6 5.7 5.0 49.2

The binding of the chimeric antigen increases stepwise with its concentration. Incubation of cells with high concentrations of murine Fcγ fragments inhibited this binding, whereas mannan (an inhibitor of CD206 receptor binding) had only a marginal effect. Therefore, CD32 may be the main receptor in the binding and uptake of this chimeric antigen.

M. Example 13: Glycosylation of the HBV S1/S2 antigen provides immunogenicity

The pathway of protein glycosylation in insect cells differs from that in mammalian cells because the glycosylation undergone by synthesized proteins in insect cells results in higher mannose content and lack of terminal sialic acid residues in secreted proteins (Altman, et al., Glycoconjug 16:109-123 (1999)). HBV S1/S2 (the antigenic component of the chimeric antigen) was expressed in both E. coli (no glycosylation) and High Five ^™ insect cells (mannose glycosylation).

1. Effect of glycosylation on antigen binding

Binding of these antigens to dendritic cells was compared as described in Example 9. Mature dendritic cells were loaded with 10 μg/mL HBVS1/S2 expressed in insect cells or E. coli. Glycosylated proteins showed better binding to dendritic cells (Table 15).

Table 15: Effect of glycosylation on HBV S1/S2 binding Specific positive cells % Relative MFI insect cells 69.9 40.3 Escherichia coli 12.2 3.9

2. The effect of glycosylation on triggering immune response

Glycosylation of HBV S1/S2 triggers enhanced immunogenicity and T cell responses. Comparison of T cell responses of HBV S1/S2 expressed in E. coli and High Five ^TM insect cells when presented by dendritic cells. Intracellular and secreted interferon-γ levels were measured as described in Example 8 (using 2.5 μg/M HBV S1/S2 protein) and the results are shown in Table 16.

Table 16: Effect of Glycosylation on Interferon-γ Levels Intracellular IFNγ (% of T cells positive for IFNγ) Secreted IFNγ(pg/ml) Baculovirus HBV S1/S2 2.1 18.9 Escherichia coli HBV S1/S2 0.83 4.3 no antigen 0.77 4.4 T cells only 0.21 1.6

HBV S1/S2 expressed in insect cells produced higher levels of intracellular and secreted interferon compared to the unglycosylated protein expressed in E. coli.

All publications and patents mentioned in the above specification are hereby incorporated by reference. Various modifications and changes will be apparent to those skilled in the art in the method and system of the present invention without departing from the spirit and scope of the present invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for carrying out the invention will be obvious to those skilled in the art, and these are within the scope of the claims of the invention.

Sequence Listing

17506.006wo1 seq listing.ST25(1).txt

SEQUENCE LISTING

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George, Rajan

Tyrrell, Lorne

Noujaim, Antoine

Wang, Dakun

Ma, Allan

<120>CHIMERIC ANTIGENS FOR BREAKING HOST TOLERANCE TO FOREIGN ANTIGENS

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<151>2004-08-08

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<400>8

agtcattctg cggccgcgag ttcgtcacag ggtccccgg 39

<210>9

<211>34

<212>DNA

17506.006wo1 seq listing.ST25(1).txt

<213>artificial sequence

<220>

<223> primer sequence

<400>9

gataaggatc ctatgggagg ttggtcatca aaac 34

<210>10

<211>40

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>10

gtcatactgc ggccgcgaaa tgtataccca gagacaaaag 40

<210>11

<211>31

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>11

tgcgctacca tggacattga ccctataaa g 31

<210>12

<211>42

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>12

tgtcattctg cggccgcgaa cattgagatt cccgagattg ag 42

<210>13

<211>40

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>13

tgtcattcag cggccgcgaa ctcttgtaaa aaagagcaga 40

<210>14

<211>40

<212>DNA

<213>artificial sequence

<220>

17506.006wo1 seq listing.ST25(1).txt

<223> primer sequence

<400>14

tgtcattctg cggccgcgtt ttcttcttca agggggggagt 40

<210>15

<211>33

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>15

tgcgctacca tggatatcaa tgcttctaga gcc 33

<210>16

<211>43

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>16

tgtcattctg cggccgcgat ttcctaggcg agggatct atg 43

<210>17

<211>27

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>17

cggaattcat gagcacgaat cctaaac 27

<210>18

<211>38

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>18

ggactagtcc ggctgaagcg ggcacagtca ggcaagag 38

<210>19

<211>27

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>19

17506.006wo1 seq listing.ST25(1).txt

ggactagtcc gaagatagag aaagagc 27

<210>20

<211>27

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>20

ccggaattct ccggttcctg gctaagg 27

<210>21

<211>28

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>21

ggactagtcc gcacacgaca tcttccgt 28

<210>22

<211>28

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>22

ccggaattct accaagtgcg caattcct 28

<210>23

<211>32

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>23

ggactagtcc ttccgcgtcg acgccggcaa at 32

<210>24

<211>32

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>24

gcggaattca cccacgtcac cgggggaaat gc 32

17506.006wo1 seq listing.ST25(1).txt

<210>25

<211>35

<212>DNA

<213>artificial sequence

<220>

<223> primer sequence

<400>25

ggactagtcc agccgcctcc gcttgggata tgagt 35

<210>26

<211>1359

<212>DNA

<213>Artificial

<220>

<223>HBV plus TBD

<220>

<221> CDS

<222>(1)..(1359)

<400>26

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat cct atg aaa aaa tgg 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Met Lys Lys Trp

20 25 30

tca tca aaa cct cgc aaa ggc atg ggg acg aat ctt tct gtt ccc aac 144

Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn

35 40 45

cct ctg gga ttc ttt ccc gat cat cag ttg gac cct gta ttc gga gcc 192

Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Val Phe Gly Ala

50 55 60

aac tca aac aat cca gat tgg gac ttc aac ccc atc aag gac cac tgg 240

Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp

65 70 75 80

cca gca gcc aac cag gta gga gtg gga gca ttc ggg cca ggg ttc acc 288

Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Phe Thr

85 90 95

cct cca cac ggc ggt gtt ttg ggg tgg agc cct cag gct cag ggc atg 336

Pro Pro His Gly Gly Val Leu Gly Trp Ser Pro Gln Ala Gln Gly Met

100 105 110

ttg acc cca gtg tca aca att cct cct cct gcc tcc gcc aat cgg cag 384

Leu Thr Pro Val Ser Thr Ile Pro Pro Pro Ala Ser Ala Asn Arg Gln

115 120 125

tca gga agg cag cct act ccc atc tct cca cct cta aga gac agt cat 432

Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His

130 135 140

cct cag gcc atg cag tgg aat tcc act gcc ttc cac caa gct ctg caa 480

17506.006wo1 seq listing.ST25(1).txt

Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln

145 150 155 160

gac ccc aga gtc agg ggt ctg tat ttt cct gct ggt ggc tcc agt tca 528

Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser

165 170 175

gga aca gta aac cct gct ccg aat att gcc tct cac atc tcg tca atc 576

Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile

180 185 190

tcc gcg agg acc ggg gac cct gtg acg aac tcg cgg ccg caa ggc ggc 624

Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Ser Arg Pro Gln Gly Gly

195 200 205

gga tcc gtg gac aag aaa att gtg ccc agg gat tgt ggt tgt aag cct 672

Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

tac ata tgt aca gtc cca gaa gta tca tct gtc ttc atc ttc ccc cca 720

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

aag ccc aag gat gtg ctc acc att act ctg act cct aag gtc acg tgt 768

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

gtt gtg gta gac atc agc aag gat gat ccc gag gtc cag ttc agc tgg 816

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

ttt gta gat gat gtg gag gtg cac aca gct cag acg caa ccc cgg gag 864

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

gag Cag ttc aac agc act ttc cgc tca gtc agt gaa ctt ccc atc atg 912

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

cac cag gac tgg ctc aat ggc aag gag ttc aaa tgc agg gtc aac agt 960

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

gca gct ttc cct gcc ccc atc gag aaa acc atc tcc aaa acc aaa ggc 1008

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

aga ccg aag gct cca cag gtg tac acc att cca cct ccc aag gag cag 1056

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

atg gcc aag gat aaa gtc agt ctg acc tgc atg ata aca gac ttc ttc 1104

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

cct gaa gac att act gtg gag tgg cag tgg aat ggg cag cca gcg gag 1152

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

aac tac aag aac act cag ccc atc atg gac aca gat ggc tct tac ttc 1200

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

17506.006wo1 seq listing.ST25(1).txt

gtc tac agc aag ctc aat gtg cag aag agc aac tgg gag gca gga aat 1248

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

act ttc acc tgc tct gtg tta cat gag ggc ctg cac aac cac cat act 1296

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

gag aag agc ctc tcc cac tct cct ggg ctg caa agc ttg tcg aga agt 1344

Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg Ser

435 440 445

act aga gga tca taa 1359

Thr Arg Gly Ser

450

<210>27

<211>452

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>27

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Met Lys Lys Trp

20 25 30

Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn

35 40 45

Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Val Phe Gly Ala

50 55 60

Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp

65 70 75 80

Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Phe Thr

85 90 95

Pro Pro His Gly Gly Val Leu Gly Trp Ser Pro Gln Ala Gln Gly Met

100 105 110

Leu Thr Pro Val Ser Thr Ile Pro Pro Pro Ala Ser Ala Asn Arg Gln

115 120 125

Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His

130 135 140

17506.006wo1 seq listing.ST25(1).txt

Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln

145 150 155 160

Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser

165 170 175

Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile

180 185 190

Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Ser Arg Pro Gln Gly Gly

195 200 205

Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

17506.006wo1 seq listing.ST25(1).txt

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg Ser

435 440 445

Thr Arg Gly Ser

450

<210>28

<211>2037

<212>DNA

<213>Artificial

<220>

<223>Hepatitis B Virus plus murine

<220>

<221> CDS

<222>(1)..(2037)

<400>28

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat cct atg gga ggt tgg 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Met Gly Gly Trp

20 25 30

tca tca aaa cct cgc aaa ggc atg ggg acg aat ctt tct gtt ccc aac 144

Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn

35 40 45

cct ctg gga ttc ttt ccc gat cat cag ttg gac cct gta ttc gga gcc 192

Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Val Phe Gly Ala

50 55 60

aac tca aac aat cca gat tgg gac ttc aac ccc atc aag gac cac tgg 240

Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp

65 70 75 80

cca gca gcc aac cag gta gga gtg gga gca ttc ggg cca ggg ttc acc 288

Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Phe Thr

85 90 95

cct cca cac ggc ggt gtt ttg ggg tgg agc cct cag gct cag ggc atg 336

Pro Pro His Gly Gly Val Leu Gly Trp Ser Pro Gln Ala Gln Gly Met

100 105 110

ttg acc cca gtg tca aca att cct cct cct gcc tcc gcc aat cgg cag 384

Leu Thr Pro Val Ser Thr Ile Pro Pro Pro Ala Ser Ala Asn Arg Gln

115 120 125

17506.006wo1 seq listing.ST25(1).txt

tca gga agg cag cct act ccc atc tct cca cct cta aga gac agt cat 432

Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His

130 135 140

cct cag gcc atg cag tgg aat tcc act gcc ttc cac caa gct ctg caa 480

Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Pne His Gln Ala Leu Gln

145 150 155 160

gac ccc aga gtc agg ggt ctg tat ttt cct gct ggt ggc tcc agt tca 528

Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser

165 170 175

gga aca gta aac cct gct ccg aat att gcc tct cac atc tcg tca atc 576

Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile

180 185 190

tcc gcg agg act ggg gac cct gtg acg aac atg gag aac atc aca tca 624

Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Met Glu Asn Ile Thr Ser

195 200 205

gga ttc cta gga ccc ctg ctc gtg tta cag gcg ggg ttt ttc ttg ttg 672

Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu

210 215 220

aca aga atc ctc aca ata ccg cag agt cta gac tcg tgg tgg act tct 720

Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser

225 230 235 240

ctc aat ttt cta ggg gga tca ccc gtg tgt ctt ggc caa aat tcg cag 768

Leu Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln

245 250 255

tcc cca acc tcc aat cac tca cca acc tcc tgt cct cca att tgt cct 816

Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro

260 265 270

ggt tat cgc tgg atg tgt ctg cgg cgt ttt atc ata ttc ctc ttc atc 864

Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile

275 280 285

ctg ctg cta tgc ctc atc ttc tta ttg gtt ctt ctg gat tat caa ggt 912

Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly

290 295 300

atg ttg ccc gtt tgt cct cta att cca gga tca aca aca acc agt acg 960

Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr

305 310 315 320

gga cca tgc aaa acc tgc acg act cct gct caa ggc aac tct atg ttt 1008

Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe

325 330 335

ccc tca tgt tgc tgt aca aaa cct acg gat gga aat tgc acc tgt att 1056

Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile

340 345 350

ccc atc cca tcg tct tgg gct ttc gca aaa tac cta tgg gag tgg gcc 1104

Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala

355 360 365

tca gtc cgt ttc tct tgg ctc agt tta cta gtg cca ttt gtt cag tgg 1152

Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp

370 375 380

17506.006wo1 seq listing.ST25(1).txt

ttc gta ggg ctt tcc ccc act gtt tgg ctt tca gct ata tgg atg atg 1200

Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met

385 390 395 400

tgg tat tgg ggg cca agt ctg tac agc atc gtg agt ccc ttt ata ccg 1248

Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro

405 410 415

ctg tta cca att ttc ttt tgt ctc tgg gta tac att tcg cgg ccg caa 1296

Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile Ser Arg Pro Gln

420 425 430

ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat tgt ggt tgt 1344

Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

435 440 445

aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc ttc atc ttc 1392

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

450 455 460

ccc cca aag ccc aag gat gtg ctc acc att act ctg act cct aag gtc 1440

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

465 470 475 480

acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag gtc cag ttc 1488

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

485 490 495

agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag acg caa ccc 1536

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

500 505 510

cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt gaa ctt ccc 1584

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

515 520 525

atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa tgc agg gtc 1632

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

530 535 540

aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc tcc aaa acc 1680

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

545 550 555 560

aaa ggc aga ccg aag gct cca cag gtg tac acc att cca cct ccc aag 1728

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

565 570 575

gag cag atg gcc aag gat aaa gtc agt ctg acc tgc atg ata aca gac 1776

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

580 585 590

ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat ggg cag cca 1824

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

595 600 605

gcg gag aac tac aag aac act cag ccc atc atg gac aca gat ggc tct 1872

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

610 615 620

tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac tgg gag gca 1920

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

17506.006wo1 seq listing.ST25(1).txt

625 630 635 640

gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg cac aac cac 1968

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

645 650 655

cat act gag aag ag agc ctc tcc cac tct cct ggg ctg caa agc ttg tcg 2016

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser

660 665 670

aga agt act aga gga tca taa 2037

Arg Ser Thr Arg Gly Ser

675

<210>29

<211>678

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>29

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Met Gly Gly Trp

20 25 30

Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn

35 40 45

Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Val Phe Gly Ala

50 55 60

Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp

65 70 75 80

Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Phe Thr

85 90 95

Pro Pro His Gly Gly Val Leu Gly Trp Ser Pro Gln Ala Gln Gly Met

100 105 110

Leu Thr Pro Val Ser Thr Ile Pro Pro Pro Ala Ser Ala Asn Arg Gln

115 120 125

Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His

130 135 140

Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln

145 150 155 160

17506.006wo1 seq listing.ST25(1).txt

Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser

165 170 175

Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile

180 185 190

Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Met Glu Asn Ile Thr Ser

195 200 205

Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu

210 215 220

Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser

225 230 235 240

Leu Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln

245 250 255

Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro

260 265 270

Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile

275 280 285

Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly

290 295 300

Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr

305 310 315 320

Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe

325 330 335

Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile

340 345 350

Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala

355 360 365

Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp

370 375 380

Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met

385 390 395 400

Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro

17506.006wo1 seq listing.ST25(1).txt

405 410 415

Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile Ser Arg Pro Gln

420 425 430

Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

435 440 445

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Tle Phe

450 455 460

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

465 470 475 480

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

485 490 495

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

500 505 510

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

515 520 525

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

530 535 540

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

545 550 555 560

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

565 570 575

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

580 585 590

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

595 600 605

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

610 615 620

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

625 630 635 640

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

645 650 655

17506.006wo1 seq listing.ST25(1).txt

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser

660 665 670

Arg Ser Thr Arg Gly Ser

675

<210>30

<211>1383

<212>DNA

<213>Artificial

<220>

<223>Hepatitis B Virus plus murine

<220>

<221> CDS

<222>(1)..(1383)

<400>30

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gac att gac cct tat aaa 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Ile Asp Pro Tyr Lys

20 25 30

gaa ttt gga gct act gtg gag tta ctc tcg ttt ttg cct tct gac ttc 144

Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe

35 40 45

ttt cct tcc gtc aga gat ctc cta gac acc gcc tcg gct ctg tat cgg 192

Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg

50 55 60

gaa gcc tta gag tct cct gag cat tgc tca cct cac cat acc gca ctc 240

Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu

65 70 75 80

agg caa gcc att ctc tgc tgg ggg gaa ttg atg act cta gct acc tgg 288

Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp

85 90 95

gtg ggt aat aat ttg gaa gat cca gca tcc agg gat cta gta gtc aat 336

Val Gly Asn Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu Val Val Asn

100 105 110

tat gtt aat act aac atg gga tta aag atc agg caa ctc ttg tgg ttt 384

Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe

115 120 125

cat atc tct tgc ctt act ttt gga aga gaa act gta ctt gaa tat ttg 432

His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu

130 135 140

gtc tct ttc gga gtg tgg att cgc act cct cca gcc tat aga cca cca 480

Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro

145 150 155 160

aat gcc cct atc tta tca aca ctt ccg gaa act act gtt gtt aga cga 528

17506.006wo1 seq listing.ST25(1).txt

Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg

165 170 175

cgg gac cga ggc agg tcc cct aga aga aga act ccc tcg cct cgc aga 576

Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg

180 185 190

cgc aga tct caa tcg ccg cgt cgc aga aga tct caa tct cgg gaa tct 624

Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser

195 200 205

caa tgt tcg cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg 672

Gln Cys Ser Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val

210 215 220

ccc agg gat tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta 720

Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val

225 230 235 240

tca tct gtc ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att 768

Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile

245 250 255

act ctg act cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat 816

Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp

260 265 270

gat ccc gag gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac 864

Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His

275 280 285

aca gct cag acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc 912

Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

290 295 300

tca gtc agt gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag 960

Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

gag ttc aaa tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag 1008

Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu

325 330 335

aaa acc atc tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac 1056

Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr

340 345 350

acc att cca cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg 1104

Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu

355 360 365

acc tgc atg ata aca gac ttc ttc cct gaa gac att act gtg gag tgg 1152

Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp

370 375 380

cag tgg aat ggg cag cca gcg gag aac tac aag aac act cag ccc atc 1200

Gln Trp Asn Gly Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile

385 390 395 400

atg gac aca gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag 1248

Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln

405 410 415

17506.006wo1 seq listing.ST25(1).txt

aag agc aac tgg gag gca gga aat act ttc acc tgc tct gtg tta cat 1296

Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His

420 425 430

gag ggc ctg cac aac cac cat act gag aag agc ctc tcc cac tct cct 1344

Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro

435 440 445

ggg ctg caa agc ttg tcg aga agt act aga gga tca taa 1383

Gly Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

450 455 460

<210>31

<211>460

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>31

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Ile Asp Pro Tyr Lys

20 25 30

Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe

35 40 45

Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg

50 55 60

Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu

65 70 75 80

Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp

85 90 95

Val Gly Asn Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu Val Val Asn

100 105 110

Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe

115 120 125

His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu

130 135 140

Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro

145 150 155 160

17506.006wo1 seq listing.ST25(1).txt

Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg

165 170 175

Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg

180 185 190

Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser

195 200 205

Gln Cys Ser Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val

210 215 220

Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val

225 230 235 240

Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile

245 250 255

Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp

260 265 270

Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His

275 280 285

Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

290 295 300

Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr

340 345 350

Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu

355 360 365

Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp

370 375 380

Gln Trp Asn Gly Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile

385 390 395 400

Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln

405 410 415

17506.006wo1 seq listing.ST25(1).txt

Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His

420 425 430

Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro

435 440 445

Gly Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

450 455 460

<210>32

<211>1326

<212>DNA

<213>Artificial

<220>

<223>Duck hepatitis B virus plus murine

<220>

<221> CDS

<222>(1)..(1326)

<400>32

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc atg ggg 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Gly

20 25 30

caa cat cca gca aaa tca atg gac gtc aga cgg ata gaa gga gga gaa 144

Gln His Pro Ala Lys Ser Met Asp Val Arg Arg Ile Glu Gly Gly Glu

35 40 45

ata ctg tta aac caa ctt gcc gga agg atg atc cca aaa ggg act ttg 192

Ile Leu Leu Asn Gln Leu Ala Gly Arg Met Ile Pro Lys Gly Thr Leu

50 55 60

aca tgg tca ggc aag ttt cca aca cta gat cac gtg tta gac cat gtg 240

Thr Trp Ser Gly Lys Phe Pro Thr Leu Asp His Val Leu Asp His Val

65 70 75 80

caa aca atg gag gag ata aac acc ctc cag aat cag gga gct tgg cct 288

Gln Thr Met Glu Glu Ile Asn Thr Leu Gln Asn Gln Gly Ala Trp Pro

85 90 95

gct gcg gcg gga agg aga gta gga tta tca aat ccg act cct caa gag 336

Ala Gly Ala Gly Arg Arg Val Gly Leu Ser Asn Pro Thr Pro Gln Glu

100 105 110

att cct cag ccc cag tgg act ccc gag gaa gac caa aaa gca cgc gaa 384

Ile Pro Gln Pro Gln Trp Thr Pro Glu Glu Asp Gln Lys Ala Arg Glu

115 120 125

gct ttt cgc cgt tat caa gaa gaa aga cca ccg gaa acc acc acc att 432

Ala Phe Arg Arg Tyr Gln Glu Glu Arg Pro Pro Glu Thr Thr Thr Ile

130 135 140

17506.006wo1 seq listing.ST25(1).txt

cct ccg tct tcc cct cct cag tgg aag cta caa ccc ggg gac gat cca 480

Pro Pro Ser Ser Pro Pro Gln Trp Lys Leu Gln Pro Gly Asp Asp Pro

145 150 155 160

ctc ctg gga aat cag tct ctc ctc gag act cat ccg cta tac cag tca 528

Leu Leu Gly Asn Gln Ser Leu Leu Glu Thr His Pro Leu Tyr Gln Ser

165 170 175

gaa cca gcg gtg cca gtg ata aaa act ccc ccc ttg aag aag aaa acg 576

Glu Pro Ala Val Pro Val Ile Lys Thr Pro Pro Leu Lys Lys Lys Thr

180 185 190

cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat 624

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

195 200 205

tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc 672

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

210 215 220

ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act ctg act 720

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

225 230 235 240

cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag 768

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

245 250 255

gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag 816

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

260 265 270

acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt 864

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

275 280 285

gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa 912

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

290 295 300

tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc 960

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

305 310 315 320

tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc att cca 1008

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

325 330 335

cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc tgc atg 1056

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

340 345 350

ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat 1104

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

355 360 365

ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg gac aca 1152

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

370 375 380

gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac 1200

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

385 390 395 400

17506.006wo1 seq listing.ST25(1).txt

tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg 1248

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

405 410 415

cac aac cac cat act gag aag agc ctc tcc cac tct cct ggg ctg caa 1296

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

420 425 430

agc ttg tcg aga agt act aga gga tca taa 1326

Ser Leu Ser Arg Ser Thr Arg Gly Ser

435 440

<210>33

<211>441

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>33

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Gly

20 25 30

Gln His Pro Ala Lys Ser Met Asp Val Arg Arg Ile Glu Gly Gly Glu

35 40 45

Ile Leu Leu Asn Gln Leu Ala Gly Arg Met Ile Pro Lys Gly Thr Leu

50 55 60

Thr Trp Ser Gly Lys Phe Pro Thr Leu Asp His Val Leu Asp His Val

65 70 75 80

Gln Thr Met Glu Glu Ile Asn Thr Leu Gln Asn Gln Gly Ala Trp Pro

85 90 95

Ala Gly Ala Gly Arg Arg Val Gly Leu Ser Asn Pro Thr Pro Gln Glu

100 105 110

Ile Pro Gln Pro Gln Trp Thr Pro Glu Glu Asp Gln Lys Ala Arg Glu

115 120 125

Ala Phe Arg Arg Tyr Gln Glu Glu Arg Pro Pro Glu Thr Thr Thr Ile

130 135 140

Pro Pro Ser Ser Pro Pro Gln Trp Lys Leu Gln Pro Gly Asp Asp Pro

145 150 155 160

17506.006wo1 seq listing.ST25(1).txt

Leu Leu Gly Asn Gln Ser Leu Leu Glu Thr His Pro Leu Tyr Gln Ser

165 170 175

Glu Pro Ala Val Pro Val Ile Lys Thr Pro Pro Leu Lys Lys Lys Thr

180 185 190

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

195 200 205

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

210 215 220

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

225 230 235 240

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

245 250 255

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

260 265 270

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

275 280 285

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

290 295 300

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

305 310 315 320

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

325 330 335

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

340 345 350

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

355 360 365

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

370 375 380

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

385 390 395 400

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

405 410 415

17506.006wo1 seq listing.ST25(1).txt

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

420 425 430

Ser Leu Ser Arg Ser Thr Arg Gly Ser

435 440

<210>34

<211>1827

<212>DNA

<213>Artificial

<220>

<223>Duck hepatitis B virus plus murine

<220>

<221> CDS

<222>(1)..(1824)

<400>34

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc atg ggg 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Gly

20 25 30

caa cat cca gca aaa tca atg gac gtc aga cgg ata gaa gga gga gaa 144

Gln His Pro Ala Lys Ser Met Asp Val Arg Arg Ile Glu Gly Gly Glu

35 40 45

ata ctg tta aac caa ctt gcc gga agg atg atc cca aaa ggg act ttg 192

Ile Leu Leu Asn Gln Leu Ala Gly Arg Met Ile Pro Lys Gly Thr Leu

50 55 60

aca tgg tca ggc aag ttt cca aca cta gat cac gtg tta gac cat gtg 240

Thr Trp Ser Gly Lys Phe Pro Thr Leu Asp His Val Leu Asp His Val

65 70 75 80

caa aca atg gag gag ata aac acc ctc cag aat cag gga gct tgg cct 288

Gln Thr Met Glu Glu Ile Asn Thr Leu Gln Asn Gln Gly Ala Trp Pro

85 90 95

gct ggg gcg gga agg aga gta gga tta tca aat ccg act cct caa gag 336

Ala Gly Ala Gly Arg Arg Val Gly Leu Ser Asn Pro Thr Pro Gln Glu

100 105 110

att cct caa ccc cag tgg act ccc gag gaa gac caa aaa gca cgc gaa 384

Ile Pro Gln Pro Gln Trp Thr Pro Glu Glu Asp Gln Lys Ala Arg Glu

115 120 125

gct ttt cgc cgt tat caa gaa gaa aga cca ccg gaa acc acc acc att 432

Ala Phe Arg Arg Tyr Gln Glu Glu Arg Pro Pro Glu Thr Thr Thr Ile

130 135 140

cct ccg tct tcc cct cct cag tgg aag cta caa ccc ggg gac gat cca 480

Pro Pro Ser Ser Pro Pro Gln Trp Lys Leu Gln Pro Gly Asp Asp Pro

145 150 155 160

17506.006wo1 seq listing.ST25(1).txt

ctc ctg gga aat cag tct ctc ctc gag act cat ccg cta tac cag tca 528

Leu Leu Gly Asn Gln Ser Leu Leu Glu Thr His Pro Leu Tyr Gln Ser

165 170 175

gaa cca gcg gtg cca gtg ata aaa act ccc ccc ttg aag aag aaa atg 576

Glu Pro Ala Val Pro Val Ile Lys Thr Pro Pro Leu Lys Lys Lys Met

180 185 190

tct ggt acc ttc ggg gga ata cta gct ggc cta atc gga tta ctg gta 624

Ser Gly Thr Phe Gly Gly Ile Leu Ala Gly Leu Ile Gly Leu Leu Val

195 200 205

agc ttt ttc ttg ttg ata aaa att cta gaa ata ctg agg agg cta gat 672

Ser Phe Phe Leu Leu Ile Lys Ile Leu Glu Ile Leu Arg Arg Leu Asp

210 215 220

tgg tgg tgg att tct ctc agt tct cca aag gga aaa atg caa tgc gct 720

Trp Trp Trp Ile Ser Leu Ser Ser Ser Pro Lys Gly Lys Met Gln Cys Ala

225 230 235 240

ttc caa gat act gga gcc caa atc tct cca cat tac gta gga tct tgc 768

Phe Gln Asp Thr Gly Ala Gln Ile Ser Pro His Tyr Val Gly Ser Cys

245 250 255

ccg tgg gga tgc cca gga ttt ctt tgg acc tat ctc agg ctt ttt atc 816

Pro Trp Gly Cys Pro Gly Phe Leu Trp Thr Tyr Leu Arg Leu Phe Ile

260 265 270

atc ttc ctc tta atc ctg cta gta gca gca ggc ttg ctg tat ctg acg 864

Ile Phe Leu Leu Ile Leu Leu Val Ala Ala Gly Leu Leu Tyr Leu Thr

275 280 285

gac aac ggg tct act att tta gga aag ctc caa tgg gcg tcg gtc tca 912

Asp Asn Gly Ser Thr Ile Leu Gly Lys Leu Gln Trp Ala Ser Val Ser

290 295 300

gcc ctt ttc tcc tcc atc tct tca cta ctg ccc tcg gat ccg aaa tct 960

Ala Leu Phe Ser Ser Ile Ser Ser Leu Leu Pro Ser Asp Pro Lys Ser

305 310 315 320

ctc gtc gct tta acg ttt gga ctt tca ctt ata tgg atg act tcc tcc 1008

Leu Val Ala Leu Thr Phe Gly Leu Ser Leu Ile Trp Met Thr Ser Ser

325 330 335

tct gcc acc caa acg ctc gtc acc tta acg caa tta gcc acg ctg tct 1056

Ser Ala Thr Gln Thr Leu Val Thr Leu Thr Gln Leu Ala Thr Leu Ser

340 345 350

gct ctt ttt tac aag agt tcg cgg ccg caa ggc ggc gga tcc gtg gac 1104

Ala Leu Phe Tyr Lys Ser Ser Arg Pro Gln Gly Gly Gly Ser Val Asp

355 360 365

aag aaa att gtg ccc agg gat tgt ggt tgt aag cct tgc ata tgt aca 1152

Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr

370 375 380

gtc cca gaa gta tca tct gtc ttc atc ttc ccc cca aag ccc aag gat 1200

Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp

385 390 395 400

gtg ctc acc att act ctg act cct aag gtc acg tgt gtt gtg gta gac 1248

Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp

17506.006wo1 seq listing.ST25(1).txt

405 410 415

atc agc aag gat gat ccc gag gtc cag ttc agc tgg ttt gta gat gat 1296

Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp

420 425 430

gtg gag gtg cac aca gct cag acg caa ccc cgg gag gag cag ttc aac 1344

Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn

435 440 445

agc act ttc cgc tca gtc agt gaa ctt ccc atc atg cac cag gac tgg 1392

Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp

450 455 460

ctc aat ggc aag gag ttc aaa tgc agg gtc aac agt gca gct ttc cct 1440

Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro

465 470 475 480

gcc ccc atc gag aaa acc atc tcc aaa acc aaa ggc aga ccg aag gct 1488

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala

485 490 495

cca cag gtg tac acc att cca cct ccc aag gag cag atg gcc aag gat 1536

Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp

500 505 510

aaa gtc agt ctg acc tgc atg ata aca gac ttc ttc cct gaa gac att 1584

Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile

515 520 525

act gtg gag tgg cag tgg aat ggg cag cca gcg gag aac tac aag aac 1632

Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn ryr Lys Asn

530 535 540

act cag ccc atc atg gac aca gat ggc tct tac ttc gtc tac agc aag 1680

Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys

545 550 555 560

ctc aat gtg cag aag agc aac tgg gag gca gga aat act ttc acc tgc 1728

Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys

565 570 575

tct gtg tta cat gag ggc ctg cac aac cac cat act gag aag agc ctc 1776

Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu

580 585 590

tcc cac tct cct ggg ctg caa agc ttg tcg aga agt act aga gga tca 1824

Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

595 600 605

taa 1827

<210>35

<211>608

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>35

17506.006wo1 seq listing.ST25(1).txt

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Gly

20 25 30

Gln His Pro Ala Lys Ser Met Asp Val Arg Arg Ile Glu Gly Gly Glu

35 40 45

Ile Leu Leu Asn Gln Leu Ala Gly Arg Met Ile Pro Lys Gly Thr Leu

50 55 60

Thr Trp Ser Gly Lys Phe Pro Thr Leu Asp His Val Leu Asp His Val

65 70 75 80

Gln Thr Met Glu Glu Ile Asn Thr Leu Gln Asn Gln Gly Ala Trp Pro

85 90 95

Ala Gly Ala Gly Arg Arg Val Gly Leu Ser Asn Pro Thr Pro Gln Glu

100 105 110

Ile Pro Gln Pro Gln Trp Thr Pro Glu Glu Asp Gln Lys Ala Arg Glu

115 120 125

Ala Phe Arg Arg Tyr Gln Glu Glu Arg Pro Pro Glu Thr Thr Thr Ile

130 135 140

Pro Pro Ser Ser Pro Pro Gln Trp Lys Leu Gln Pro Gly Asp Asp Pro

145 150 155 160

Leu Leu Gly Asn Gln Ser Leu Leu Glu Thr His Pro Leu Tyr Gln Ser

165 170 175

Glu Pro Ala Val Pro Val Ile Lys Thr Pro Pro Leu Lys Lys Lys Met

180 185 190

Ser Gly Thr Phe Gly Gly Ile Leu Als Gly Leu Ile Gly Leu Leu Val

195 200 205

Ser Phe Phe Leu Leu Ile Lys Ile Leu Glu Ile Leu Arg Arg Leu Asp

210 215 220

Trp Trp Trp Ile Ser Leu Ser Ser Ser Pro Lys Gly Lys Met Gln Cys Ala

225 230 235 240

Phe Gln Asp Thr Gly Ala Gln Ile Ser Pro His Tyr Val Gly Ser Cys

245 250 255

17506.006wo1 seq listing.ST25(1).txt

Pro Trp Gly Cys Pro Gly Phe Leu Trp Thr Tyr Leu Arg Leu Phe Ile

260 265 270

Ile Phe Leu Leu Ile Leu Leu Val Ala Ala Gly Leu Leu Tyr Leu Thr

275 280 285

Asp Asn Gly Ser Thr Ile Leu Gly Lys Leu Gln Trp Ala Ser Val Ser

290 295 300

Ala Leu Phe Ser Ser Ile Ser Ser Leu Leu Pro Ser Asp Pro Lys Ser

305 310 315 320

Leu Val Ala Leu Thr Phe Gly Leu Ser Leu Ile Trp Met Thr Ser Ser

325 330 335

Ser Ala Thr Gln Thr Leu Val Thr Leu Thr Gln Leu Ala Thr Leu Ser

340 345 350

Ala Leu Phe Tyr Lys Ser Ser Arg Pro Gln Gly Gly Gly Ser Val Asp

355 360 365

Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr

370 375 380

Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp

385 390 395 400

Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp

405 410 415

Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp

420 425 430

Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn

435 440 445

Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp

450 455 460

Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro

465 470 475 480

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala

485 490 495

Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp

500 505 510

17506.006wo1 seq listing.ST25(1).txt

Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile

515 520 525

Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn

530 535 540

Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys

545 550 555 560

Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys

565 570 575

Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu

580 585 590

Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

595 600 605

<210>36

<211>1614

<212>DNA

<213>Artificial

<220>

<223>Duck hepatitis B virus plus murine

<220>

<221> CDS

<222>(1)..(1614)

<400>36

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat atc aat gct tct aga 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Ile Asn Ala Ser Arg

20 25 30

gcc tta gcc aat gtg tat gat cta cca gat gat ttc ttt cca aaa ata 144

Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro Lys Ile

35 40 45

gat gat ctt gtt aga gat gct aaa gac gct tta gag cct tat tgg aaa 192

Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr Trp Lys

50 55 60

tca gat tca ata aag aaa cat gtt ttg att gca act cac ttt gtg gat 240

Ser Asp Ser Ile Lys Lys His Val Leu Ile Ala Thr His Phe Val Asp

65 70 75 80

ctc att gaa gac ttc tgg cag act aca cag ggc atg cat gaa ata gcc 288

Leu Ile Glu Asp Phe Trp Gln Thr Thr Gln Gly Met His Glu Ile Ala

85 90 95

17506.006wo1 seq listing.ST25(1).txt

gaa tca tta aga gct gtt ata cct ccc act act act cct gtt cca ccg 336

Glu Ser Leu Arg Ala Val Ile Pro Pro Thr Thr Thr Pro Val Pro Pro

100 105 110

ggt tat ctt att cag cac gag gaa gct gaa gag ata cct ttg gga gat 384

Gly Tyr Leu Ile Gln His Glu Glu Ala Glu Glu Ile Pro Leu Gly Asp

115 120 125

tta ttt aaa cac caa gaa gaa agg ata gta agt ttc caa ccc gac tat 432

Leu Phe Lys His Gln Glu Glu Arg Ile Val Ser Phe Gln Pro Asp Tyr

130 135 140

ccg att acg gct aga att cat gct cat ttg aaa gct tat gca aaa att 480

Pro Ile Thr Ala Arg Ile His Ala His Leu Lys Ala Tyr Ala Lys Ile

145 150 155 160

aac gag gaa tca ctg gat agg gct agg aga ttg ctt tgg tgg cat tac 528

Asn Glu Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp His Tyr

165 170 175

aac tgt tta ctg tgg gga gaa gct caa gtt act aac tat att tct cgt 576

Asn Cys Leu Leu Trp Gly Glu Ala Gln Val Thr Asn Tyr Ile Ser Arg

180 185 190

ttg cgt act tgg ttg tca act cct gag aaa tat aga ggt aga gat gcc 624

Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Arg Asp Ala

195 200 205

ccg acc att gaa gca atc act aga cca atc cag gtg gct cag gga ggc 672

Pro Thr Ile Glu Ala Ile Thr Arg Pro Ile Gln Val Ala Gln Gly Gly

210 215 220

aga aaa aca act acg ggt act aga aaa cct cgt gga ctc gaa cct aga 720

Arg Lys Thr Thr Thr Gly Thr Arg Lys Pro Arg Gly Leu Glu Pro Arg

225 230 235 240

aga aga aaa gtt aaa acc aca gtt gtc tat ggg aga aga cgt tca aag 768

Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg Ser Lys

245 250 255

tcc cgg gaa agg aga gcc cct aca ccc caa cgt gcg ggc tcc cct ctc 816

Ser Arg Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala Gly Ser Pro Leu

260 265 270

cca cgt agt agt tcg agc agc cac cat aga tct ccc tcg cct agg aaa tcg 864

Pro Arg Ser Ser Ser Ser His His Arg Ser Pro Ser Pro Arg Lys Ser

275 280 285

cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat 912

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

290 295 300

tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc 960

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

305 310 315 320

ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act ctg act 1008

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

325 330 335

cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag 1056

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

17506.006wo1 seq listing.ST25(1).txt

340 345 350

gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag 1104

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

355 360 365

acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt 1152

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

370 375 380

gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa 1200

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

385 390 395 400

tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc 1248

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

405 410 415

tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc att cca 1296

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

420 425 430

cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc tgc atg 1344

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

435 440 445

ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat 1392

lle Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

450 455 460

ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg gac aca 1440

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

465 470 475 480

gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac 1488

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

485 490 495

tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg 1536

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

500 505 510

cac aac cac cat act gag aag agc ctc tcc cac tct cct ggg ctg caa 1584

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

515 520 525

agc ttg tcg aga agt act aga gga tca taa 1614

Ser Leu Ser Arg Ser Thr Arg Gly Ser

530 535

<210>37

<211>537

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>37

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

17506.006wo1 seq listing.ST25(1).txt

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Ile Asn Ala Ser Arg

20 25 30

Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro Lys Ile

35 40 45

Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr Trp Lys

50 55 60

Ser Asp Ser Ile Lys Lys His Val Leu Ile Ala Thr His Phe Val Asp

65 70 75 80

Leu Ile Glu Asp Phe Trp Gln Thr Thr Gln Gly Met His Glu Ile Ala

85 90 95

Glu Ser Leu Arg Ala Val Ile Pro Pro Thr Thr Thr Pro Val Pro Pro

100 105 110

Gly Tyr Leu Ile Gln His Glu Glu Ala Glu Glu Ile Pro Leu Gly Asp

115 120 125

Leu Phe Lys His Gln Glu Glu Arg Ile Val Ser Phe Gln Pro Asp Tyr

130 135 140

Pro Ile Thr Ala Arg Ile His Ala His Leu Lys Ala Tyr Ala Lys Ile

145 150 155 160

Asn Glu Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp His Tyr

165 170 175

Asn Cys Leu Leu Trp Gly Glu Ala Gln Val Thr Asn Tyr Ile Ser Arg

180 185 190

Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Arg Asp Ala

195 200 205

Pro Thr Ile Glu Ala Ile Thr Arg Pro Ile Gln Val Ala Gln Gly Gly

210 215 220

Arg Lys Thr Thr Thr Gly Thr Arg Lys Pro Arg Gly Leu Glu Pro Arg

225 230 235 240

Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg Ser Lys

245 250 255

Ser Arg Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala Gly Ser Pro Leu

17506.006wo1 seq listing.ST25(1).txt

260 265 270

Pro Arg Ser Ser Ser Ser His His Arg Ser Pro Ser Pro Arg Lys Ser

275 280 285

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

290 295 300

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

305 310 315 320

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

325 330 335

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

340 345 350

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

355 360 365

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

370 375 380

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

385 390 395 400

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

405 410 415

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

420 425 430

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

435 440 445

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

450 455 460

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

465 470 475 480

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

485 490 495

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

500 505 510

17506.006wo1 seq listing.ST25(1).txt

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

515 520 525

Ser Leu Ser Arg Ser Thr Arg Gly Ser

530 535

<210>38

<211>1422

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

<220>

<221> CDS

<222>(1)..(1422)

<400>38

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc atg agc 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Ser

20 25 30

acg aat cct aaa cct caa aga aaa acc aaa cgt aac acc aac cgt cgc 144

Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

35 40 45

cca cag gac gtc aag ttc ccg ggt ggc ggt cag atc gtt ggt gga gtt 192

Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val

50 55 60

tac ttg ttg ccg cgc agg ggc cct aga ttg ggt gtg cgc gcg acg agg 240

Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg

65 70 75 80

aag act tcc gag cgg tcg caa cct cga ggt aga cgt cag cct atc ccc 288

Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro

85 90 95

aag gca cgt cgg ccc gag ggc agg acc tgg gct cag ccc ggg tac cct 336

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro

100 105 110

tgg ccc ctc tat ggc aat gag ggt tgc ggg tgg gcg gga tgg ctc ctg 384

Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu

115 120 125

tct ccc cgt ggc tct cgg cct agc tgg ggc ccc aca gac ccc cgg cgt 432

Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg

130 135 140

agg tcg cgc aat ttg ggt aag gtc atc gat acc ctt acg tgc ggc ttc 480

Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe

145 150 155 160

gcc gac ctc atg ggg tac ata ccg ctc gtc ggc gcc cct ctt gga ggc 528

17506.006wo1 seq listing.ST25(1).txt

Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly

165 170 175

gct gcc agg gcc ctg gcg cat ggc gtc cgg gtt ctg gaa gac ggc gtg 576

Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val

180 185 190

aac tat gca aca ggg aac ctt cct ggt tgc tct ttc tct atc ttc ctt 624

Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu

195 200 205

ctg gcc ctg ctc tct tgc ctg act gtg ccc gct tca gcc gga cta gtg 672

Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Gly Leu Val

210 215 220

cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat 720

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

225 230 235 240

tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc 768

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

245 250 255

ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act ctg act 816

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

260 265 270

cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag 864

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

275 280 285

gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag 912

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

290 295 300

acg caa ccc ccg gag gag cag ttc aac agc act ttc cgc tca gtc agt 960

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

305 310 315 320

gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa 1008

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

325 330 335

tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc 1056

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

340 345 350

tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc att cca 1104

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

355 360 365

cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc tgc atg 1152

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

370 375 380

ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat 1200

Tle Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

385 390 395 400

ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg gac aca 1248

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

405 410 415

17506.006wo1 seq listing.ST25(1).txt

gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac 1296

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

420 425 430

tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg 1344

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

435 440 445

cac aac cac cat act gag aag agc ctc tcc cac tct cct ggg ctg caa 1392

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

450 455 460

agc ttg tcg aga agt act aga gga tca taa 1422

Ser Leu Ser Arg Ser Thr Arg Gly Ser

465 470

<210>39

<211>473

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>39

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Ser

20 25 30

Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

35 40 45

Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val

50 55 60

Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg

65 70 75 80

Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro

85 90 95

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro

100 105 110

Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu

115 120 125

Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg

130 135 140

17506.006wo1 seq listing.ST25(1).txt

Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe

145 150 155 160

Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly

165 170 175

Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val

180 185 190

Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu

195 200 205

Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Gly Leu Val

210 215 220

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

225 230 235 240

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

245 250 255

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

260 265 270

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

275 280 285

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

290 295 300

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

305 310 315 320

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

325 330 335

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

340 345 350

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

355 360 365

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

370 375 380

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

385 390 395 400

17506.006wo1 seq listing.ST25(1).txt

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

405 410 415

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

420 425 430

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

435 440 445

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

450 455 460

Ser Leu Ser Arg Ser Thr Arg Gly Ser

465 470

<210>40

<211>1380

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

<220>

<221> CDS

<222>(1)..(1380)

<400>40

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc atg agc 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Ser

20 25 30

acg aat cct aaa cct caa aga aaa acc aaa cgt aac acc aac cgt cgc 144

Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Ash Thr Asn Arg Arg

35 40 45

cca cag gac gtc aag ttc ccg ggt ggc ggt cag atc gtt ggt gga gtt 192

Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val

50 55 60

tac ttg ttg ccg cgc agg ggc cct aga ttg ggt gtg cgc gcg acg agg 240

Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg

65 70 75 80

aag act tcc gag cgg tcg caa cct cga ggt aga cgt cag cct atc ccc 288

Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro

85 90 95

aag gca cgt cgg ccc gag ggc agg acc tgg gct cag ccc ggg tac cct 336

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro

100 105 110

17506.006wo1 seq listing.ST25(1).txt

tgg ccc ctc tat ggc aat gag ggt tgc ggg tgg gcg gga tgg ctc ctg 384

Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu

115 120 125

tct ccc cgt ggc tct cgg cct agc tgg ggc ccc aca gac ccc cgg cgt 432

Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg

130 135 140

agg tcg cgc aat ttg ggt aag gtc atc gat acc ctt acg tgc ggc ttc 480

Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe

145 150 155 160

gcc gac ctc atg ggg tac ata ccg ctc gtc ggc gcc cct ctt gga ggc 528

Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly

165 170 175

gct gcc agg gcc ctg gcg cat ggc gtc cgg gtt ctg gaa gac ggc gtg 576

Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val

180 185 190

aac tat gca aca ggg aac ctt cct ggt tgc tct ttc tct atc ttc gga 624

Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Gly

195 200 205

cta gtg cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc 672

Leu Val Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro

210 215 220

agg gat tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca 720

Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser

225 230 235 240

tct gtc ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act 768

Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr

245 250 255

ctg act cct aag gtc acg tgt gtt gtg gta gac atc aag gat gat 816

Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp

260 265 270

ccc gag gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca 864

Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr

275 280 285

gct cag acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca 912

Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser

290 295 300

gtc agt gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag 960

Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

ttc aaa tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa 1008

Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys

325 330 335

acc atc tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc 1056

Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr

340 345 350

att cca cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc 1104

Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr

355 360 365

17506.006wo1 seq listing.ST25(1).txt

tgc atg ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag 1152

Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln

370 375 380

tgg aat ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg 1200

Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met

385 390 395 400

gac aca gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag 1248

Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys

405 410 415

agc aac tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag 1296

Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu

420 425 430

ggc ctg cac aac cac cat act gag aag ag agc ctc tcc cac tct cct ggg 1344

Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440 445

ctg caa agc ttg tcg aga agt act aga gga tca taa 1380

Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

450 455

<210>41

<211>459

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>41

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Met Ser

20 25 30

Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

35 40 45

Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val

50 55 60

Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg

65 70 75 80

Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro

85 90 95

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro

100 105 110

17506.006wo1 seq listing.ST25(1).txt

Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu

115 120 125

Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg

130 135 140

Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe

145 150 155 160

Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly

165 170 175

Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val

180 185 190

Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Gly

195 200 205

Leu Val Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro

210 215 220

Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser

225 230 235 240

Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr

245 250 255

Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp

260 265 270

Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr

275 280 285

Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser

290 295 300

Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr

340 345 350

Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr

355 360 365

17506.006wo1 seq listing.ST25(1).txt

Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln

370 375 380

Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met

385 390 395 400

Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys

405 410 415

Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu

420 425 430

Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440 445

Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

450 455

<210>42

<211>2190

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

<220>

<221> CDS

<222>(1)..(2190)

<400>42

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc tcc ggt 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Ser Gly

20 25 30

tcc tgg cta agg gac atc tgg gac tgg ata tgc gag gtg ctg agc gac 144

Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp

35 40 45

ttt aag acc tgg ctg aaa gcc aag ctc atg cca caa ctg cct ggg att 192

Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile

50 55 60

ccc ttt gtg tcc tgc cag cgc ggg tat agg ggg gtc tgg cga gga gac 240

Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp

65 70 75 80

ggc att atg cac act cgc tgc cac tgt gga gct gag atc act gga cat 288

Gly Ile Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His

85 90 95

17506.006wo1 seq listing.ST25(1).txt

gtc aaa aac ggg acg atg agg atc gtc ggt cct agg acc tgc agg aac 336

Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn

100 105 110

atg tgg agt ggg acg ttc ccc att aac gcc tac acc acg ggc ccc tgt 384

Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys

115 120 125

act ccc ctt cct gcg ccg aac tat aag ttc gcg ctg tgg agg gtg tct 432

Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser

130 135 140

gca gag gaa tac gtg gag ata agg cgg gtg ggg gac ttc cac tac gta 480

Ala Glu Glu Tyr Val Glu Ile Arg Arg Val Gly Asp Phe His Tyr Val

145 150 155 160

tcg ggt atg act act gac aat ctt aaa tgc ccg tgc cag atc cca tcg 528

Ser Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser

165 170 175

ccc gaa ttt ttc aca gaa ttg gac ggg gtg cgc cta cac agg ttt gcg 576

Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala

180 185 190

ccc cct tgc aag ccc ttg ctg cgg gag gag gta tca ttc aga gta gga 624

Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly

195 200 205

ctc cac gag tac ccg gtg ggg tcg caa tta cct tgc gag ccc gaa ccg 672

Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro

210 215 220

gac gta gcc gtg ttg acg tcc atg ctc act gat ccc tcc cat ata aca 720

Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr

225 230 235 240

gca gag gcg gcc ggg aga agg ttg gcg aga ggg tca ccc cct tct atg 768

Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Met

245 250 255

gcc agc tcc tcg gct agc cag ctg tcc gct cca tct ctc aag gca act 816

Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr

260 265 270

tgc acc gcc aac cat gac tcc cct gac gac gag ctc ata gag gct aac 864

Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn

275 280 285

ctc ctg tgg agg cag gag atg ggc ggc aac atc acc agg gtt gag tca 912

Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser

290 295 300

gag aac aaa gtg gtg att ctg gac tcc ttc gat ccg ctt gtg gca gag 960

Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu

305 310 315 320

gag gat gag cgg gag gtc tcc gta cct gca gaa att ctg cgg aag tct 1008

Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Lys Ser

325 330 335

cgg aga ttc gcc cgg gcc ctg ccc gtc tgg gcg cgg ccg gac tac aac 1056

Arg Arg Phe Ala Arg Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn

17506.006wo1 seq listing.ST25(1).txt

340 345 350

ccc ccg cta gta gag acg tgg aaa aag cct gac tac gaa cca cct gtg 1104

Pro Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val

355 360 365

gtc cat ggc tgc ccg cta cca cct cca cgg tcc cct cct gtg cct ccg 1152

Val His Gly Cys Pro Leu Pro Pro Pro Arg Ser Pro Pro Val Pro Pro

370 375 380

cct cgg aaa aag cgt acg gtg gtc ctc acc gaa tca acc cta tct act 1200

Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr

385 390 395 400

gcc ttg gcc gag ctt gcc acc aaa agt ttt ggc agc tcc tca act tcc 1248

Ala Leu Ala Glu Leu Ala Thr Lys Ser Phe Gly Ser Ser Ser Thr Ser

405 410 415

ggc att acg ggc gac aat acg aca aca tcc tct gag ccc gcc cct tct 1296

Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser

420 425 430

ggc tgc ccc ccc gac tcc gac gtt gag tcc tat tct tcc atg ccc ccc 1344

Gly Cys Pro Pro Asp Ser Asp Val Glu Ser Tyr Ser Ser Met Pro Pro

435 440 445

ctg gag ggg gag cct ggg gat ccg gat ctc agc gac ggg tca tgg tcg 1392

Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser

450 455 460

acg gtc agt agt ggg gcc gac acg gaa gat gtc gtg tgc gga cta gtg 1440

Thr Val Ser Ser Gly Ala Asp Thr Glu Asp Val Val Cys Gly Leu Val

465 470 475 480

cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat 1488

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

485 490 495

tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc 1536

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

500 505 510

ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act ctg act 1584

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

515 520 525

cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag 1632

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

530 535 540

gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag 1680

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

545 550 555 560

acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt 1728

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

565 570 575

gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa 1776

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

580 585 590

tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc 1824

17506.006wo1 seq listing.ST25(1).txt

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

595 600 605

tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc att cca 1872

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

610 615 620

cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc tgc atg 1920

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

625 630 635 640

ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat 1968

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

645 650 655

ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg gac aca 2016

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

660 665 670

gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac 2064

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

675 680 685

tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg 2112

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

690 695 700

cac aac cac cat act gag aag agc ctc tcc cac tct cct ggg ctg caa 2160

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

705 710 715 720

agc ttg tcg aga agt act aga gga tca taa 2190

Ser Leu Ser Arg Ser Thr Arg Gly Ser

725

<210>43

<210>729

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>43

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Ser Gly

20 25 30

Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp

35 40 45

Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile

50 55 60

Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp

17506.006wo1 seq listing.ST25(1).txt

65 70 75 80

Gly Ile Met His Thr Arg Cys His Cys Gly Ala Glu Tle Thr Gly His

85 90 95

Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn

100 105 110

Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys

115 120 125

Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser

130 135 140

Ala Glu Glu Tyr Val Glu Ile Arg Arg Val Gly Asp Phe His Tyr Val

145 150 155 160

Ser Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser

165 170 175

Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala

180 185 190

Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly

195 200 205

Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro

210 215 220

Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr

225 230 235 240

Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Met

245 250 255

Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr

260 265 270

Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn

275 280 285

Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser

290 295 300

Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu

305 310 315 320

17506.006wo1 seq listing.ST25(1).txt

Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Lys Ser

325 330 335

Arg Arg Phe Ala Arg Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn

340 345 350

Pro Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val

355 360 365

Val His Gly Cys Pro Leu Pro Pro Pro Arg Ser Pro Pro Val Pro Pro

370 375 380

Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr

385 390 395 400

Ala Leu Ala Glu Leu Ala Thr Lys Ser Phe Gly Ser Ser Ser Thr Ser

405 410 415

Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser

420 425 430

Gly Cys Pro Pro Asp Ser Asp Val Glu Ser Tyr Ser Ser Met Pro Pro

435 440 445

Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser

450 455 460

Thr Val Ser Ser Gly Ala Asp Thr Glu Asp Val Val Cys Gly Leu Val

465 470 475 480

Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp

485 490 495

Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val

500 505 510

Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr

515 520 525

Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu

530 535 540

Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln

545 550 555 560

Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser

565 570 575

17506.006wo1 seq listing.ST25(1).txt

Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys

580 585 590

Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile

595 600 605

Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro

610 615 620

Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met

625 630 635 640

Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn

645 650 655

Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr

660 665 670

Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn

675 680 685

Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu

690 695 700

His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln

705 710 715 720

Ser Leu Ser Arg Ser Thr Arg Gly Ser

725

<210>44

<211>1428

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

<220>

<221> CDS

<222>(1)..(1428)

<400>44

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc tac caa 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Tyr Gln

20 25 30

17506.006wo1 seq listing.ST25(1).txt

gtg cgc aat tcc tcg ggg ctt tac cat gtc acc aat gat tgc cct aac 144

Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn

35 40 45

tcg agt att gtg tac gag gcg gcc gat gcc atc ctg cac act ccg ggg 192

Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly

50 55 60

tgt gtc cct tgc gtt cgc gag ggt aac gcc tcg agg tgt tgg gtg gcg 240

Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala

65 70 75 80

gtg acc ccc acg gtg gcc acc agg gac ggc aaa ctc ccc aca acg cag 288

Val Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln

85 90 95

ctt cga cgt cat atc gat ctg ctt gtc ggg agc gcc acc ctc tgc tcg 336

Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser

100 105 110

gcc ctc tac gtg ggg gac ctg tgc ggg tct gtc ttt ctt gtt ggt caa 384

Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln

115 120 125

ctg ttt acc ttc tct ccc agg cgc cac tgg acg acg caa gac tgc aat 432

Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Asp Cys Asn

130 135 140

tgt tct atc tat ccc ggc cat ata acg ggt cat cgc atg gca tgg gat 480

Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp

145 150 155 160

atg atg atg aac tgg tcc cct acg gca gcg ttg gtg gta gct cag ctg 528

Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu

165 170 175

ctc cgg atc cca caa gcc atc atg gac atg atc gct ggt gct cac tgg 576

Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile Ala Gly Ala His Trp

180 185 190

gga gtc ctg gcg ggc ata gcg tat ttc tcc atg gtg ggg aac tgg gcg 624

Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala

195 200 205

aag gtc ctg gta gtg ctg ctg cta ttt gcc ggc gtc gac gcg gaa gga 672

Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Gly

210 215 220

cta gtg cgg ccg caa ggc ggc gga tcc gtg gac aag aaa att gtg ccc 720

Leu Val Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro

225 230 235 240

agg gat tgt ggt tgt aag cct tgc ata tgt aca gtc cca gaa gta tca 768

Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser

245 250 255

tct gtc ttc atc ttc ccc cca aag ccc aag gat gtg ctc acc att act 816

Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr

260 265 270

ctg act cct aag gtc acg tgt gtt gtg gta gac atc agc aag gat gat 864

Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp

275 280 285

17506.006wo1 seq listing.ST25(1).txt

ccc gag gtc cag ttc agc tgg ttt gta gat gat gtg gag gtg cac aca 912

Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr

290 295 300

gct cag acg caa ccc cgg gag gag cag ttc aac agc act ttc cgc tca 960

Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser

305 310 315 320

gtc agt gaa ctt ccc atc atg cac cag gac tgg ctc aat ggc aag gag 1008

Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

ttc aaa tgc agg gtc aac agt gca gct ttc cct gcc ccc atc gag aaa 1056

Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys

340 345 350

acc atc tcc aaa acc aaa ggc aga ccg aag gct cca cag gtg tac acc 1104

Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr

355 360 365

att cca cct ccc aag gag cag atg gcc aag gat aaa gtc agt ctg acc 1152

Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr

370 375 380

tgc atg ata aca gac ttc ttc cct gaa gac att act gtg gag tgg cag 1200

Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln

385 390 395 400

tgg aat ggg cag cca gcg gag aac tac aag aac act cag ccc atc atg 1248

Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met

405 410 415

gac aca gat ggc tct tac ttc gtc tac agc aag ctc aat gtg cag aag 1296

Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys

420 425 430

agc aac tgg gag gca gga aat act ttc acc tgc tct gtg tta cat gag 1344

Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu

435 440 445

ggc ctg cac aac cac cat act gag aag agc ctc tcc cac tct cct ggg 1392

Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

450 455 460

ctg caa agc ttg tcg aga agt act aga gga tca taa 1428

Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

465 470 475

<210>45

<211>475

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>45

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

17506.006wo1 seq listing.ST25(1).txt

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Tyr Gln

20 25 30

Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn

35 40 45

Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly

50 55 60

Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala

65 70 75 80

Val Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln

85 90 95

Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser

100 105 110

Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln

115 120 125

Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Asp Cys Asn

130 135 140

Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp

145 150 155 160

Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu

165 170 175

Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile Ala Gly Ala His Trp

180 185 190

Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala

195 200 205

Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Gly

210 215 220

Leu Val Arg Pro Gln Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro

225 230 235 240

Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser

245 250 255

Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr

260 265 270

17506.006wo1 seq listing.ST25(1).txt

Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp

275 280 285

Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr

290 295 300

Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser

305 310 315 320

Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr

355 360 365

Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr

370 375 380

Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln

385 390 395 400

Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met

405 410 415

Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys

420 425 430

Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu

435 440 445

Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

450 455 460

Leu Gln Ser Leu Ser Arg Ser Thr Arg Gly Ser

465 470 475

<210>46

<211>1938

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

17506.006wo1 seq listing.ST25(1).txt

<220>

<221> CDS

<222>(1)..(1938)

<400>46

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc acc cac 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Thr His

20 25 30

gtc acc ggg gga aat gcc ggc cgc acc acg gct ggg ctt gtt ggt ctc 144

Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu Val Gly Leu

35 40 45

ctt aca cca ggc gcc aag cag aac atc caa ctg atc aac acc aac ggc 192

Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly

50 55 60

agt tgg cac atc aat agc acg gcc ttg aat tgc aat gaa agc ctt aac 240

Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn

65 70 75 80

acc ggc tgg tta gca ggg ctc ttc tat caa cac aaa ttc aac tct tca 288

Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser Ser

85 90 95

ggc tgt cct gag agg ttg gcc agc tgc cga cgc ctt acc gat ttt gcc 336

Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Ala

100 105 110

cag ggc tgg ggt cct atc agt tat gcc aac gga agc ggc ctc gac gaa 384

Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu

115 120 125

cgc ccc tac tgc tgg cac tac cct cca aga cct tgt ggc att gtg ccc 432

Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro

130 135 140

gca aag agc gtg tgt ggc ccg gta tat tgc ttc act ccc agc ccc gtg 480

Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val

145 150 155 160

gtg gtg gga acg acc gac agg tcg ggc gcg cct acc tac agc tgg ggt 528

Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly

165 170 175

gca aat gat acg gat gtc ttc gtc ctt aac aac acc agg cca ccg ctg 576

Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu

180 185 190

ggc aat tgg ttc ggt tgt acc tgg atg aac tca act gga ttc acc aaa 624

Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys

195 200 205

gtg tgc gga gcg ccc cct tgt gtc atc gga ggg gtg ggc aac aac acc 672

Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn Thr

210 215 220

ttg ctc tgc ccc act gat tgc ttc cgc aaa cat ccg gaa gcc aca tac 720

Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr

17506.006wo1 seq listing.ST25(1).txt

225 230 235 240

tct cgg tgc ggc tcc ggt ccc tgg att aca ccc agg tgc atg gtc gac 768

Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Met Val Asp

245 250 255

tac ccg tat agg ctt tgg cac tat cct tgt acc atc aat tac acc ata 816

Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile

260 265 270

ttc aaa gtc agg atg tac gtg gga ggg gtc gag cac agg ctg gaa gcg 864

Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala

275 280 285

gcc tgc aac tgg acg cgg ggc gaa cgc tgt gat ctg gaa gac agg gac 912

Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp

290 295 300

agg tcc gag ctc agc ccg ttg ctg ctg tcc acc aca cag tgg cag gtc 960

Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln Val

305 310 315 320

ctt ccg tgt tct ttc acg acc ctg cca gcc ttg tcc acc ggc ctc atc 1008

Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile

325 330 335

cac ctc cac cag aac att gtg gac gtg cag tac ttg tac ggg gta ggg 1056

His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly

340 345 350

tca agc atc gcg tcc tgg gcc att aag tgg gag tac gtc gtt ctc ctg 1104

Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu

355 360 365

ttc ctt ctg ctt gca gac gcg cgc gtc tgc tcc tgc ttg tgg atg atg 1152

Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met Met

370 375 380

tta ctc ata tcc caa gcg gag gcg gct gga cta gtg cgg ccg caa ggc 1200

Leu Leu Ile Ser Gln Ala Glu Ala Ala Gly Leu Val Arg Pro Gln Gly

385 390 395 400

ggc gga tcc gtg gac aag aaa att gtg ccc agg gat tgt ggt tgt aag 1248

Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

405 410 415

cct tgc ata tgt aca gtc cca gaa gta tca tct gtc ttc atc ttc ccc 1296

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

420 425 430

cca aag ccc aag gat gtg ctc acc att act ctg act cct aag gtc acg 1344

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

435 440 445

tgt gtt gtg gta gac atc agc aag cat gat ccc gag gtc cag ttc agc 1392

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

450 455 460

tgg ttt gta gat gat gtg gag gtg cac aca gct cag acg caa ccc cgg 1440

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

465 470 475 480

gag gag cag ttc aac agc act ttc cgc tca gtc agt gaa ctt ccc atc 1488

17506.006wo1 seq listing.ST25(1).txt

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

485 490 495

atg cac cag gac tgg ctc aat ggc aag gag ttc aaa tgc agg gtc aac 1536

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

500 505 510

agt gca gct ttc cct gcc ccc atc gag aaa acc atc tcc aaa acc aaa 1584

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

515 520 525

ggc aga ccg aag gct cca cag gtg tac acc att cca cct ccc aag gag 1632

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

530 535 540

cag atg gcc aag gat aaa gtc agt ctg acc tgc atg ata aca gac ttc 1680

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

545 550 555 560

ttc cct gaa gac att act gtg gag tgg cag tgg aat ggg cag cca gcg 1728

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

565 570 575

gag aac tac aag aac act cag ccc atc atg gac aca gat ggc tct tac 1776

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

580 585 590

ttc gtc tac agc aag ctc aat gtg cag aag agc aac tgg gag gca gga 1824

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

595 600 605

aat act ttc acc tgc tct gtg tta cat gag ggc ctg cac aac cac cat 1872

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

610 615 620

act gag aag agc ctc tcc cac tct cct ggg ctg caa agc ttg tcg aga 1920

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg

625 630 635 640

agt act aga gga tca taa 1938

Ser Thr Arg Gly Ser

645

<210>47

<211>645

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>47

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asr Lau Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Thr His

20 25 30

Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu Val Gly Leu

17506.006wo1 seq listing.ST25(1).txt

35 40 45

Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly

50 55 60

Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn

65 70 75 80

Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser Ser

85 90 95

Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Ala

100 105 110

Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu

115 120 125

Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro

130 135 140

Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val

145 150 155 160

Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly

165 170 175

Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu

180 185 190

Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys

195 200 205

Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn Thr

210 215 220

Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr

225 230 235 240

Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Met Val Asp

245 250 255

Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile

260 265 270

Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala

275 280 285

17506.006wo1 seq listing.ST25(1).txt

Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp

290 295 300

Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln Val

305 310 315 320

Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile

325 330 335

His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly

340 345 350

Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu

355 360 365

Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met Met

370 375 380

Leu Leu Ile Ser Gln Ala Glu Ala Ala Gly Leu Val Arg Pro Gln Gly

385 390 395 400

Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

405 410 415

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

420 425 430

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

435 440 445

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

450 455 460

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

465 470 475 480

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

485 490 495

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

500 505 510

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

515 520 525

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

530 535 540

17506.006wo1 seq listing.ST25(1).txt

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

545 550 555 560

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

565 570 575

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

580 585 590

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

595 600 605

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

610 615 620

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser Arg

625 630 635 640

Ser Thr Arg Gly Ser

645

<210>48

<211>2517

<212>DNA

<213>Artificial

<220>

<223>Hepatitis C virus plus murine

<220>

<221> CDS

<222>(1)..(2517)

<400>48

atg tcg tac tac cat cac cat cac cat cac gat tac gat atc cca acg 48

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttc tac caa 96

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Tyr Gln

20 25 30

gtg cgc aat tcc tcg ggg ctt tac cat gtc acc aat gat tgc cct aac 144

Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn

35 40 45

tcg agt att gtg tac gag gcg gcc gat gcc atc ctg cac act ccg ggg 192

Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly

50 55 60

tgt gtc cct tgc gtt cgc gag ggt aac gcc tcg agg tgt tgg gtg gcg 240

Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala

65 70 75 80

17506.006wo1 seq listing.ST25(1).txt

gtg acc ccc acg gtg gcc acc agg gac ggc aaa ctc ccc aca acg cag 288

Val Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln

85 90 95

ctt cga cgt cat atc gat ctg ctt gtc ggg agc gcc acc ctc tgc tcg 336

Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser

100 105 110

gcc ctc tac gtg ggg gac ctg tgc ggg tct gtc ttt ctt gtt ggt caa 384

Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln

115 120 125

ctg ttt acc ttc tct ccc agg cgc cac tgg acg acg caa gac tgc aat 432

Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Asp Cys Asn

130 135 140

tgt tct atc tat ccc ggc cat ata acg ggt cat cgc atg gca tgg gat 480

Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp

145 150 155 160

atg atg atg aac tgg tcc cct acg gca gcg ttg gtg gta gct cag ctg 528

Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu

165 170 175

ctc cgg atc cca caa gcc atc atg gac atg atc gct ggt gct cac tgg 576

Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile Ala Gly Ala His Trp

180 185 190

gga gtc ctg gcg ggc ata gcg tat ttc tcc atg gtg ggg aac tgg gcg 624

Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala

195 200 205

aag gtc ctg gta gtg ctg ctg cta ttt gcc ggc gtc gac gcg gaa acc 672

Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr

210 215 220

cac gtc acc ggg gga aat gcc ggc cgc acc acg gct ggg ctt gtt ggt 720

His Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu Val Gly

225 230 235 240

ctc ctt aca cca ggc gcc aag cag aac atc caa ctg atc aac acc aac 768

Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn

245 250 255

ggc agt tgg cac atc aat agc acg gcc ttg aat tgc aat gaa agc ctt 816

Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu

260 265 270

aac acc ggc tgg tta gca ggg ctc ttc tat caa cac aaa ttc aac tct 864

Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser

275 280 285

tca ggc tgt cct gag agg ttg gcc agc tgc cga cgc ctt acc gat ttt 912

Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe

290 295 300

gcc cag ggc tgg ggt cct atc agt tat gcc aac gga agc ggc ctc gac 960

Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp

305 310 315 320

gaa cgc ccc tac tgc tgg cac tac cct cca aga cct tgt ggc att gtg 1008

Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val

325 330 335

17506.006wo1 seq listing.ST25(1).txt

ccc gca aag agc gtg tgt ggc ccg gta tat tgc ttc act ccc agc ccc 1056

Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro

340 345 350

gtg gtg gtg gga acg acc gac agg tcg ggc gcg cct acc tac agc tgg 1104

Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp

355 360 365

ggt gca aat gat acg gat gtc ttc gtc ctt aac aac acc agg cca ccg 1152

Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro

370 375 380

ctg ggc aat tgg ttc ggt tgt acc tgg atg aac tca act gga ttc acc 1200

Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr

385 390 395 400

aaa gtg tgc gga gcg ccc cct tgt gtc atc gga ggg gtg ggc aac aac 1248

Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn

405 410 415

acc ttg ctc tgc ccc act gat tgc ttc cgc aaa cat ccg gaa gcc aca 1296

Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr

420 425 430

tac tct cgg tgc ggc tcc ggt ccc tgg att aca ccc agg tgc atg gtc 1344

Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Met Val

435 440 445

gac tac ccg tat agg ctt tgg cac tat cct tgt acc atc aat tac acc 1392

Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr

450 455 460

ata ttc aaa gtc agg atg tac gtg gga ggg gtc gag cac agg ctg gaa 1440

Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu

465 470 475 480

gcg gcc tgc aac tgg acg cgg ggc gaa cgc tgt gat ctg gaa gac agg 1488

Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg

485 490 495

gac agg tcc gag ctc agc ccg ttg ctg ctg tcc acc aca cag tgg cag 1536

Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln

500 505 510

gtc ctt ccg tgt tct ttc acg acc ctg cca gcc ttg tcc acc ggc ctc 1584

Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu

515 520 525

atc cac ctc cac cag aac att gtg gac gtg cag tac ttg tac ggg gta 1632

Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val

530 535 540

ggg tca agc atc gcg tcc tgg gcc att aag tgg gag tac gtc gtt ctc 1680

Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu

545 550 555 560

ctg ttc ctt ctg ctt gca gac gcg cgc gtc tgc tcc tgc ttg tgg atg 1728

Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met

565 570 575

atg tta ctc ata tcc caa gcg gag gcg gct gga cta gtg cgg ccg caa 1776

Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Gly Leu Val Arg Pro Gln

17506.006wo1 seg listing.ST25(1).txt

580 585 590

ggc ggc gga tcc gtg gac aag aaa att gtg ccc agg gat tgt ggt tgt 1824

Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

595 600 605

aag cct tgc ata tgt aca gtc cca gaa gta tca tct gtc ttc atc ttc 1872

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

610 615 620

ccc cca aag ccc aag gat gtg ctc acc att act ctg act cct aag gtc 1920

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

625 630 635 640

acg tgt gtt gtg gta gac atc agc aag gat gat ccc gag gtc cag ttc 1968

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

645 650 655

agc tgg ttt gta gat gat gtg gag gtg cac aca gct cag acg caa ccc 2016

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

660 665 670

cgg gag gag cag ttc aac agc act ttc cgc tca gtc agt gaa ctt ccc 2064

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

675 680 685

atc atg cac cag gac tgg ctc aat ggc aag gag ttc aaa tgc agg gtc 2112

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

690 695 700

aac agt gca gct ttc cct gcc ccc atc gag aaa acc atc tcc aaa acc 2160

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

705 710 715 720

aaa ggc aga ccg aag gct cca cag gtg tac acc att cca cct ccc aag 2208

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

725 730 735

gac cag atg gcc aag gat aaa gtc agt ctg acc tgc atg ata aca gac 2256

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

740 745 750

ttc ttc cct gaa gac att act gtg gag tgg cag tgg aat ggg cag cca 2304

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

755 760 765

gcg gag aac tac aag aac act cag ccc atc atg gac aca gat ggc tct 2352

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

770 775 780

tac ttc gtc tac agc aag ctc aat gtg cag aag agc aac tgg gag gca 2400

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

785 790 795 800

gga aat act ttc acc tgc tct gtg tta cat gag ggc ctg cac aac cac 2448

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

805 810 815

cat act gag aag agc ctc tcc cac tct cct ggg ctg caa agc ttg tcg 2496

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser

820 825 830

aga agt act aga gga tca taa 2517

17506.006wo1 seq listing.ST25(1).txt

Arg Ser Thr Arg Gly Ser

835

<210>49

<211>838

<212>PRT

<213>Artificial

<220>

<223>Synthetic Construct

<400>49

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr

1 5 10 15

Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Tyr Gln

20 25 30

Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn

35 40 45

Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly

50 55 60

Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala

65 70 75 80

Val Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln

85 90 95

Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser

100 105 110

Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln

115 120 125

Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Asp Cys Asn

130 135 140

Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp

145 150 155 160

Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu

165 170 175

Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile Ala Gly Ala His Trp

180 185 190

Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala

17506.006wo1 seq listing.ST25(1).txt

195 200 205

Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr

210 215 220

His Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu Val Gly

225 230 235 240

Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn

245 250 255

Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu

260 265 270

Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser

275 280 285

Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe

290 295 300

Ala Gln Gly Trp Gly Pro Tle Ser Tyr Ala Asn Gly Ser Gly Leu Asp

305 310 315 320

Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val

325 330 335

Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro

340 345 350

Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp

355 360 365

Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro

370 375 380

Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr

385 390 395 400

Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn

405 410 415

Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr

420 425 430

Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Met Val

435 440 445

17506.006wo1 seq listing.ST25(1).txt

Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr

450 455 460

Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu

465 470 475 480

Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg

485 490 495

Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln

500 505 510

Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu

515 520 525

Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val

530 535 540

Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu

545 550 555 560

Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met

565 570 575

Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Gly Leu Val Arg Pro Gln

580 585 590

Gly Gly Gly Ser Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

595 600 605

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

610 615 620

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

625 630 635 640

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

645 650 655

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

660 665 670

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

675 680 685

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

690 695 700

17506.006wo1 seq listing.ST25(1).txt

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

705 710 715 720

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

725 730 735

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

740 745 750

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

755 760 765

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

770 775 780

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

785 790 795 800

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

805 810 815

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Leu Gln Ser Leu Ser

820 825 830

Arg Ser Thr Arg Gly Ser

835

<210>50

<211>9

<212>PRT

<213>Hepatitis B virus

<400>50

Gly Met Leu Thr Pro Val Ser Thr Ile

1 5

<210>51

<211>9

<212>PRT

<213>Hepatitis B virus

<400>51

Asn Ile Ala Ser His Ile Ser Ser Ile

1 5

Form PCT/RO/134

Figure 1A

Figure 1B

figure 2

image 3

Figure 4

Figure 5

Claims (77)

1. chimeric antigen that is used to cause immunne response, described chimeric antigen comprises immunne response structural domain and target binding domains, wherein said target binding domains comprises antibody fragment.

2. chimeric antigen according to claim 1, wherein said antibody fragment are the xenotype antibody fragment.

3. chimeric antigen according to claim 1, wherein said antibody fragment are non-xenotype antibody fragment.

4. chimeric antigen according to claim 1, wherein said chimeric antigen comprise the immunne response structural domain more than.

5. chimeric antigen according to claim 1, wherein said immunne response structural domain comprises proteic at least one antigen part, described albumen is selected from: HBV albumen, DHBV albumen and HCV albumen.

6. chimeric antigen according to claim 5, wherein said immunne response structural domain comprise proteic at least one antigen part of HBV, and described HBV albumen is selected from: HBVS1/S2, HBV S1/S2/S, HBV core protein, HBV core ctm and HBV polysaccharase.

7. chimeric antigen according to claim 5, wherein said immunne response structural domain comprise proteic at least one antigen part of DHBV, and described DHBV albumen is selected from: DHBV PreS/S, DHBV PreS, DHBV core protein and DHBV polysaccharase.

8. chimeric antigen according to claim 5, wherein said immunne response structural domain comprises proteic at least one antigen part of HCV, and described HCV albumen is selected from: HCV core protein (1-191), HCV core protein (1-177), HCV E1-E2, HCV E1, HCV E2, HCV NS3, HCV NS5A and HCV NS4A.

9. according to each described chimeric antigen among the claim 1-8, wherein said immunne response structural domain further comprises the 6xHis label that is fused on the described albumen.

10. according to each described chimeric antigen among the claim 1-9, wherein said immunne response structural domain comprises from the antigenic proteic one or more antigen part of HPV, HIV, HSV, obligate born of the same parents entozoa or cancer.

11. according to each described chimeric antigen among the claim 1-10, wherein said target binding domains can be attached on the antigen presenting cell.

12. according to each described chimeric antigen among the claim 1-11, wherein said antibody fragment comprises the heavy chain immunoglobulin fragment.

13. chimeric antigen according to claim 12, wherein said heavy chain immunoglobulin fragment comprises hinge area.

14. chimeric antigen according to claim 12, wherein said chimeric antigen further comprise amino acid, VDKKI (SEQ ID NO:2).

15. chimeric antigen according to claim 12, wherein said heavy chain immunoglobulin fragment comprises C _H2 and C _H3 structural domains.

16. according to each described chimeric antigen among the claim 1-15, wherein said antibody fragment is the Fc fragment.

17. according to each described chimeric antigen among the claim 1-15, wherein said antibody fragment comprises portion C _H1 district and antibody hinge region.

18., further comprise the linker that is used to connect described immunne response structural domain and described target binding domains according to each described chimeric antigen among the claim 1-17.

19. chimeric antigen according to claim 18, wherein said linker are covalency peptide linker.

20. chimeric antigen according to claim 19, wherein said peptide linker comprise sequence SRPQGGGS or VRPQGGGS (SEQ ID NO:1).

21. chimeric antigen according to claim 18, wherein said linker is selected from leucine zipper and vitamin H/avidin.

22. according to each described chimeric antigen among the claim 1-21, wherein said chimeric antigen is by the seminose glycosylation.

23. chimeric antigen according to claim 22, wherein said immunne response structural domain is by the seminose glycosylation.

24. chimeric antigen according to claim 22, wherein said target binding domains is by the seminose glycosylation.

25. chimeric antigen according to claim 22, wherein said chimeric antigen is by few seminose glycosylation.

26. chimeric antigen according to claim 22, wherein said chimeric antigen is by the high mannose glycosylation.

27. chimeric antigen according to claim 1, wherein said target binding domains comprise the C of aminoacid sequence SRPQGGGS (SEQ ID NO:1), VDKKI (SEQ IDNO:2) and rat immune globulin CH _H2 and C _H3 structural domains.

28. according to each described chimeric antigen among the claim 1-27, it causes multi-epitope and replys.

29. chimeric antigen according to claim 1, it causes the immunne response at least one epi-position of immunne response structural domain.

30. according to each described chimeric antigen among the claim 1-29, it causes humoral immunoresponse(HI).

31. according to each described chimeric antigen among the claim 1-29, its trigger cell immunne response.

32. according to each described chimeric antigen among the claim 1-29, it causes Th1 immunne response, Th2 immunne response, CTL replys or its combination.

33. one kind is improved the method that the antigen presenting cell endoantigen is presented, described method comprises antigen presenting cell is contacted with comprising the composition according to each described chimeric antigen among the claim 1-32.

34. method according to claim 33, wherein said antigen presenting cell are dendritic cell.

35. method according to claim 34, wherein said method has improved the antigen presentation more than an epi-position.

36. method according to claim 34, wherein said method has improved the antigen presentation of at least one epi-position of described immunne response structural domain.

37. an active antigen is the method for delivery cell, described method comprise with described antigen presenting cell with contact according to each described chimeric antigen among the claim 1-32.

38. according to claim 33 or 37 described methods, wherein said contact occurs in external.

39. according to claim 33 or 37 described methods, wherein said contact takes place in vivo.

40. according to the described method of claim 39, wherein said contact occurs in the human body.

41. comprising, a method that causes immunne response, described method will comprise that the composition according to each described chimeric antigen among the claim 1-32 imposes on target object.

42. a method of breaking tolerance, described method comprise with antigen presenting cell with contact according to each chimeric antigen among the claim 1-32.

43. a method that is used for the treatment of immunity-medicable disease comprises the target object that each described chimeric antigen among the claim 1-32 is imposed on needs.

44. according to each described method among the claim 41-43, applying separately of wherein said immunne response structural domain do not cause immunne response.

45. according to each described method among the claim 41-43, the immunne response that the applying of wherein said chimeric antigen caused is greater than the immunne response that is caused that applies of independent immunne response structural domain.

46. according to the described method of claim 45, wherein said immunne response is measured by the quantity that is present in the antigen-specific antibodies in the target object.

47. according to the described method of claim 45, wherein said immunne response by the T cellular response in be exposed to be loaded with described chimeric antigen or only the quantity of the secreted interferon-of the antigen presenting cell of immunne response structural domain measure.

48. according to the described method of claim 45, wherein said immunne response is by being loaded with described chimeric antigen or the only antigen presenting cell of immunne response structural domain and the antigen-specific CD8 of initiating response in response to being exposed to ⁺The quantity of T cell is measured.

49. according to each described method among the claim 41-43, wherein said target object suffers from infection or cancer.

50. according to the described method of claim 49, wherein said infection is virus infection or parasitic infection.

51. according to the described method of claim 50, wherein said infection is a chronic viral infection.

52. according to the described method of claim 51, wherein said chronic viral infection is chronic HBV infection or chronic hcv infection, chronic human papillomavirus infection, chronic HIV (human immunodeficiency virus) infection or chronic herpes simplex infections.

53. according to the described method of claim 52, wherein said infection is that hepatitis b virus infected and described immunne response structural domain comprises proteic at least one antigen part that is selected from HBV core protein, HBVS albumen, HBV S1 albumen and HBV S2 albumen and composition thereof.

54. according to the described method of claim 52, wherein said infection is that infection with hepatitis C virus and described immunne response structural domain comprise proteic at least one antigen part that is selected from HCV core (1-191) albumen, HCV core (1-177) albumen, HCV E1 albumen, HCV E2 albumen, HCV E1-E2 albumen, HCV NS3 albumen, HCV NS5A albumen and composition thereof.

55. comprising, a method that is the target object inoculating needle to the vaccine of virus infection, described method will impose on described target object according to each described chimeric antigen among the claim 1-32.

56., wherein be the vaccine of described target object therapeutic ground inoculating needle to the virus infection of existence according to the described method of claim 55.

57. according to the described method of claim 55, wherein be described target object prophylactically inoculating needle to the vaccine of the virus infection that exists.

58. according to the described method of claim 55, wherein said target object produces the immunne response more than an epi-position at described chimeric antigen.

59. according to the described method of claim 58, wherein said target object produces the immunne response more than an epi-position at described immunne response structural domain.

60. a pharmaceutical composition comprises pharmaceutically acceptable vehicle and according to each described chimeric antigen among the claim 1-32.

61. according to the described pharmaceutical composition of claim 60, wherein said pharmaceutical composition is configured to be used for parenteral and uses.

62. according to the described pharmaceutical composition of claim 60, wherein said pharmaceutical composition is configured to be used for percutaneous dosing, intradermal administration, intravenous administration, subcutaneous administration, intramuscular administration, via intranasal application administration, through lung administration or oral administration.

63. goods comprise according to each described chimeric antigen among the claim 1-32 and the specification sheets that is used for described chimeric antigen is applied to the target object of this chimeric antigen of needs.

64. a method that generates chimeric antigen comprises:

(a) provide microorganism or clone, it comprises the polynucleotide of coding according to each described chimeric antigen among the claim 1-32; And

(b) under the condition that described chimeric antigen is expressed, cultivate described microorganism or clone.

65. according to the described method of claim 64, wherein said microorganism or clone are eukaryotic microorganisms or clone.

66. according to the described method of claim 65, wherein said microorganism or clone are yeast, plant cell or insect cell line.

67. according to the described method of claim 64, wherein said chimeric antigen is translated the back and modifies to comprise the seminose glycosylation.

68. the polynucleotide of the chimeric antigen of encoding, described polynucleotide comprises first polynucleotide portion of coding immunne response structural domain and the second polynucleotide portion of coding target binding domains, and wherein said target binding domains comprises antibody fragment.

69. according to the described polynucleotide of claim 68, wherein said immunne response structural domain comprises at least one antigen part of viral protein.

70. according to the described polynucleotide of claim 69, wherein said viral protein is HBV albumen or HCV albumen.

71. according to the described polynucleotide of claim 68, wherein said antibody fragment is the xenotype antibody fragment.

72. according to the described polynucleotide of claim 68, wherein said antibody fragment is non-xenotype antibody fragment.

73. according to the described polynucleotide of claim 68, wherein said polynucleotide comprises the nucleotide sequence that is selected from following sequence: the 1st to 1326 Nucleotide of SEQ ID NO:26, the the 1st to 2004 Nucleotide of SEQ ID NO:28, the the 1st to 1350 Nucleotide of SEQ IDNO:30, the the 1st to 1293 Nucleotide of SEQ ID NO:32, the the 1st to 1794 Nucleotide of SEQ ID NO:34, the the 1st to 1581 Nucleotide of SEQID NO:36, the the 1st to 1389 Nucleotide of SEQ ID NO:38, the the 1st to 1347 Nucleotide of SEQ ID NO:40, the the 1st to 2157 Nucleotide of SEQ ID NO:42, the the 1st to 1395 Nucleotide of SEQ ID NO:44, the the 1st to 1905 Nucleotide of SEQ ID NO:46 or the 1st to 2484 Nucleotide of SEQ ID NO:48.

74. according to the described polynucleotide of claim 68, the chimeric antigen that wherein said polymerized nucleoside acid encoding is identical with the complete aminoacid sequence at least 90% that is selected from following sequence: the 1st to 442 amino acid of SEQ ID NO:27, the the 1st to 668 amino acid of SEQ ID NO:29, the the 1st to 450 amino acid of SEQ ID NO:31, the the 1st to 431 amino acid of SEQ ID NO:33, the the 1st to 598 amino acid of SEQ ID NO:35, the the 1st to 527 amino acid of SEQ ID NO:37, the the 1st to 463 amino acid of SEQ ID NO:39, the the 1st to 449 amino acid of SEQ ID NO:41, the the 1st to 719 amino acid of SEQ ID NO:43, the the 1st to 465 amino acid of SEQ ID NO:45, the the 1st to 635 amino acid of SEQ ID NO:47, or the 1st to 828 amino acid of SEQ ID NO:49.

75. according to the described polynucleotide of claim 68, wherein said polynucleotide under the condition of strictness optionally with the polymerized nucleoside acid hybridization with the nucleotide sequence that is selected from following sequence: SEQ ID NO:26,28,30,32,34,36,38,40,42,44,46 and 48.

76. comprise microorganism or clone according to the described polynucleotide of claim 68.

77. comprise carrier according to the described polynucleotide of claim 68.

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